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Activity Report 2007/08

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Page 1: Activity Report 2007/08

Tätigkeitsbericht2007/2008

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Stiftung bürgerlichen RechtsMartinistraße 52 · 20251 HamburgTel.: +49 (0) 40 480 51-0 · Fax: +49 (0) 40 480 [email protected] · www.hpi-hamburg.de

Page 2: Activity Report 2007/08

Impressum

Verantwortlich Prof. Dr. Thomas Dobner für den Inhalt Dr. Heinrich Hohenberg

Redaktion Dr. Angela Homfeld Dr. Nicole Nolting

Grafik & Layout AlsterWerk MedienService GmbH Hamburg

Druck Hartung Druck + Medien GmbH Hamburg

Titelbild

Neu gestaltete Fassade des Seuchenlaborgebäudes

Page 3: Activity Report 2007/08

Heinrich-Pette-Institutfür Experimentelle Virologie und Immunologiean der Universität Hamburg

Martinistraße 52 · 20251 HamburgPostfach 201652 · 20206 Hamburg

Telefon: +49-40/4 80 51-0Telefax: +49-40/4 80 51-103E-Mail: [email protected]: www.hpi-hamburg.de

Das Heinrich-Pette-Institut ist Mitgliedder Leibniz-Gemeinschaft (WGL)Internet: www.wgl.de

Tätigkeitsbericht2007/2008

Page 4: Activity Report 2007/08
Page 5: Activity Report 2007/08

Allgemeiner ÜberblickVorwort ................................................................................................... 1

Die Struktur des Heinrich-Pette-Instituts .............................................. 2

Modernisierung des HPI erfolgreich abgeschlossen ............................ 4

60 Jahre HPI .............................................................................................. 5

Offen für den Dialog .............................................................................. 6

Preisverleihungen und Ehrungen .......................................................... 8

Personelle Veränderungen in den Jahren 2007 und 2008 ................... 10

Wissenschaftliche Highlights ................................................................. 12

Hohes Drittmittelaufkommen am HPI ................................................... 16

HPI fördert wissenschaftlichen Nachwuchs .......................................... 19

Gleichstellungsinitiative am HPI ............................................................ 22

Das „Leibniz Center for Infection“ (LCI) ................................................ 23

Programmbereich „Virus-Wirts-Wechselwirkungen“ Program Area “Virus-Host Interaction”*

Introduction ............................................................................................. 27

Research Projects ..................................................................................... 29

■ Helper function of wild-type p53 in Simian virus 40 induced cellular transformation ........................................................................... 29

■ Role of c-Met in SV40-induced mammary tumorigenesis ....................... 30■ Mutant p53 gain of function in mammary carcinogenesis ...................... 32■ A preclinical model for monitoring treatment and prevention

of metastasis in mammary carcinoma .................................................... 33■ Induction of a cellular immune response against SV40

induced mammary carcinoma in transgenic mice ................................... 34■ Steroid dependent control of HIV replication .......................................... 35■ Control of viral mRNA export by adenovirus regulatory proteins ............ 36■ Identification of a new substrate of the adenovirus/E1B-55K/E4orf6

ubiquitin ligase complex ......................................................................... 39■ Human adenovirus E4 region orf3/4 protein is required

for efficient virus replication ................................................................... 40■ Localization and importance of the adenovirus E4orf4 protein

during lytic infection .............................................................................. 41■ Intrinsic disorder in the amino terminus of human adenovirus

type 5 E1B-55K and its related proteins ................................................. 42■ A novel 49K isoform of the adenovirus type 5 early region

1B 55K protein is sufficient to support viral replication ........................... 43■ Daxx-mediated negative regulation of adenovirus

replication is counteracted by the adenovirus E1B-55K protein .............. 43■ Posttranscriptional processing of HIV-1 mRNA ........................................ 44

Inhaltsverzeichnis

*Titel im Inhaltsverzeichnis sind z. T. gekürzt aufgelistet.

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■ Combating drug resistance in HIV-1 infection ........................................ 47■ Eradication of HIV-1 proviral DNA from patient cells .............................. 49■ Rapid response kinetics to HLA-B57/58 Gag-restricted CTL epitopes

is associated with slow disease progression in chronic HIV-1 infection .... 51■ Determinants of successful antiviral treatment of concomitant

acute HCV/HIV coinfection ..................................................................... 53■ Crossing the Species Barrier: Role of Env-Receptor Interactions

in Gammaretrovirus Infections ................................................................ 54■ Requirement of Activation for Hepatitis B Virus Infection ....................... 55■ Enzymatic treatment of duck hepatitis B virus: topology of the

surface proteins for virions and noninfectious subviral particles .............. 55■ Entry of Duck Hepatitis B Virus into Primary Duck Liver

and Kidney Cells .................................................................................... 55■ Elucidation of the SV40-induced DNA damage signaling cascades

and their components utilized by SV40 .................................................. 56■ Role of the host’s p53 status in SV40 life cycle ....................................... 57■ Elucidation of the molecular mechanisms that underlie

SV40-induced polyploidization ............................................................... 58■ Inhibition of duck hepatitis B virus infection of liver cells

by combined treatment with viral e antigen and carbohydrates ............. 59■ Solution structure of stem-loop alpha of the hepatitis B virus

post-transcriptional regulatory element .................................................. 60■ Analysis of Duck Hepatitis B Virus core protein phosphorylation

and dephosphorylation ........................................................................... 61■ Transactivation activity of the X and PreS protein of duck hepatitis B

virus are dispensible for DHBV infection in vitro and in vivo ................... 62■ Polyomavirus-encoded miRNAs .............................................................. 63■ A Global Analysis of Evolutionary Conservation Among Known

and Predicted Gammaherpesvirus microRNAs ........................................ 65■ Monitoring the Epigenetic Fate of Gammaherpesvirus Genomes ............ 67■ A requirement for Drosha in the Maintenance of Kaposi's

sarcoma-associated Herpesvirus Latency ................................................. 68■ A FACS-based FRET assay to analyze interactions between viral

and cellular proteins in living cells .......................................................... 70■ Inhibition of Transferrin-Receptor Uptake by SIV and HIV Nef-alleles ...... 71■ Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I

Correlates with Loss of CD4+ T Cells in Natural SIV Infection ................. 72■ Single Nef Proteins from HIV-1 Subtypes C and F Fail

to Up-Regulate Invariant Chain Cell Surface Expression .......................... 73■ Human Immunodeficiency Virus Type 1 Nef Expression

Prevents AP-2-Mediated Internalization of the Major Histocompatibility Complex Class II-Associated Invariant Chain .............. 73

■ Siglec expression on chimpanzee T-cells might contribute to the asymptomatic course of lentiviral infection in human primates ........ 75

■ HIV-1 budding and persistence in macrophages ..................................... 75■ A serine phosphorylation site in HIV-1 Vpu is required

for efficient counteraction of tetherin and replication in macrophages ... 77■ Viral determinants for AIDS-progression in pediatric HIV-1

subtype B and C infections .................................................................. 78

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■ Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1 Is Restricted by both PML and Sp100 ..................................................... 78

■ Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry ............................................... 79

■ Strong and selective inhibitors of hepatitis B virus replication among novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues ........... 80

■ Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles ........................................................ 81

■ Avian Hepatitis B Viruses: Molecular and Cellular Biology, Phylogenesis and Host Tropism .............................................................. 82

■ Efficiency of DHBV infection is restricted by an abortive entry pathway .. 83■ Secretion of duck hepatitis B virus occurs via exocytosis ......................... 83■ Naturally occurring adefovir resistance mutation rtI233V

selected under adefovir therapy ............................................................. 85■ Cellular Determinants of HBV Replication ............................................... 85■ High resolution electron microscopical (EM)-analysis of the distribution

of TNF receptor 1 ................................................................................... 86■ Cryotomography Ultrastructural analysis of cytomegalovirus

tegument protein interactions ................................................................ 87■ Dynamic processes in organic and biological samples

investigated with pump-probe experiments ............................................ 88■ Size and Surface Effects on the MRI Relaxivity

of Manganese Ferrite Nanoparticle Contrast Agents .............................. 89■ Real-time magnetic resonance imaging and quantification

of lipoprotein metabolism in vivo using nanocrystals .............................. 90

Programmbereich „Zelluläre Dysregulation“ Research Program “Cellular Dysregulation“ Introduction ............................................................................................. 91

Research Projects .................................................................................... 96A. Cellular defense mechanismsA1. Cellular target genes of viral transformation■ Cell transformation by human adenoviruses ........................................... 96■ RNA interference as a tool to study adenovirus

E1A/ E1B-mediated cell transformation .................................................. 98■ Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines ..... 99

A2. Apoptosis, Senescence, and Autophagy■ p53-Rb2/p130 crosstalk in cellular senescence ..................................... 101■ Dissecting the mechanism of apoptosis-resistance

in hematopoietic malignancies expressing wild-type p53 ...................... 102■ FLASH meets nuclear bodies: CD95 receptor signals

via a nuclear pathway ........................................................................... 103■ Functional p53 is required for effective telomerase inhibition

in BCR-ABL-positive CML cells in vitro ................................................. 104■ Invasin- and β1-Integrin-dependent uptake of bacteria

is coupled to the activation of autophagy ............................................ 105

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A3. Mechanisms DNA repair■ Regulation and function of the stress-induced polymerase

α-primase-wtp53 complex ................................................................... 106■ Repair fidelity of UV-induced lesions depends on ATR and

the p53 status ...................................................................................... 107■ In UV-damaged S-phase cells, delayed origin loading of

Pol α depends on the ATR-∆p53-p21-cyclin A-Cdk2 pathway .............. 108■ Dissection of the role of SPOC1 in DNA repair ..................................... 110■ A role for E1B-AP5 in ATR signalling pathways during

adenovirus infection ............................................................................. 111

B. Cellular controls of self-renewal, proliferation, and differentiationB1. Gene transcription and chromatin modulation■ Modulation of gene expression in U251 glioblastoma cells by

binding of mutant p53 R273H to intronic and intergenic sequences .... 112■ Mutant and wild-type p53 as modulator

of global chromatinorganization .......................................................... 113■ P53 as a potential epigenetic modulator of gene expression ................ 113■ Oncogenicity and expression profiling of SPOC1 in normal

and tumor tissues ................................................................................ 114■ SPOC1: A novel PHD containing protein modulating

chromatin structure and mitotic chromosome condensation ................ 115■ Enhanced SPOC1 expression induces chromatin instability ................... 116■ SPOC1: A novel chromatin remodeling protein? ................................... 117■ The RUNX1 transcription factor: A gatekeeper in acute leukaemia ....... 119

B2. Regulators of stem cells, proliferation, and differentiation■ The MADS transcription factor MEF2C is an important regulator

of monopoiesis .................................................................................... 121■ Aldehyde dehydrogenase 1 (ALDH1) as a functional marker

of cancer stem cells in a mammary carcinoma mouse model ............... 122■ Hierarchy and p53-dependent differentiation of embryonal

stem/progenitor cells in mouse F9 teratocarcinoma cell line ................. 123■ Spontaneous F9 cell differentiation in a capillary-based 3D cell culture ... 125■ SPOC1 is essential for spermatogonial stem cells ................................. 127■ Activated receptor tyrosine kinases and their downstream

pathways in acute myelogenous leukemia ........................................... 128■ Identification of genetic mutations that cooperate with constitutively

active FLT3 in the induction of acute lymphoblastic leukemia (ALL) ...... 130■ Differential regulation of human TERT gene expression and telomerase

activity during liver regeneration and liver cell differentiation ............... 132■ Regulation of human and mouse TERT promoter activity during

progression of mammary carcinogenesis .............................................. 132■ Physiological role of cellular prion protein in insulin producing β-cells .. 133

C. Molecular mechanisms for “fine tuning” normal cell processesC1. Post-transcriptional and translational control mechanisms■ Dissecting nucleocytoplasmic transport of CD83 mRNA ....................... 134

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■ Qualitative and quantitative image analysis of ultra structural changes in sarcomere assembly of cMyBP-C knock-in mice .................. 135

■ Translational Elongation of Cyclin D1 requires an RNA Chaperone ....... 136■ The fusion protein OTT-MAL induces R-loop formation

and genomic instability ........................................................................ 137

C2. Functional regulation by protein modifications■ Adenovirus type 5 early region 1 B 55-kDa oncoprotein is modified

by different isoforms of the small ubiquitin-related modifier (SUMO) ...... 138■ The adenovirus E1B-55K oncoprotein induces SUMO modification of p53 .. 138■ Characterization of posttranslational modifications of SPOC1 .............. 139■ The ubiquitin-specific protease USP7 is a target

of the adenovirus E1B-55K oncoprotein ............................................... 140■ The MDM2 ubiquitination signal in the DNA-binding domain of p53

forms a docking site for calcium calmodulin kinase superfamily members .. 141■ Homeodomain-interacting protein kinase 2 is the ionizing

radiation-activated p53 serine 46 kinase and is regulated by ATM ....... 142

Leibniz Center for Infection (LCI)■ Ultrastructural investigation of the in vitro life cycle of Schistosoma

mansoni, analysed by cryo-techniques and TEM-tomography ............... 143■ Electron microscopical dissection of the exoerythrocytic

forms of Plasmodium berghei in situ .................................................... 144

Personelle Zusammensetzung der Wissenschaftlichen Abteilungen, Forschungs- und Nachwuchsgruppen .......................... 145

Veröffentlichungen ............................................................................... 158

Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten, Bachelorarbeiten ....................................................... 166

Lehrtätigkeit .......................................................................................... 171

Gutachtertätigkeit ................................................................................ 172

Organisation von Meetings und Chairman-Tätigkeiten .................... 174

Mitherausgabe wissenschaftlicher Zeitschriften ............................... 176

Drittmittelprojekte ................................................................................ 177

Erfindungsmeldungen und Patente .................................................... 183

Finanzielle Förderung ........................................................................... 184

Seminare ................................................................................................ 186

Mitglieder des Wissenschaftlichen Beirats ......................................... 190

Mitglieder des Kuratoriums ................................................................. 191

Kaufmännische Abteilung .................................................................... 192

Personal der Kaufmännischen Abteilung und Allgemeine Dienste ... 193

Beauftragte ........................................................................................... 196

Organigramm ........................................................................................ 197

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Gemälde von Heinrich Pette. Es schmückt nun das Foyer des HPI im neuen Ersatz- und Erweiterungsbau II.

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1

VorwortDieser Tätigkeitsbericht des Heinrich-Pette-Instituts (HPI) stellt die aktuellen Entwicklungen der wissenschaftlichen Programme und der Aktivitäten der Mit-arbeiter in den Jahren 2007 und 2008 detailliert dar. Zugleich informiert das HPI mit diesem Bericht über seine wichtigsten Forschungsergebnisse, seine zentra-len Ziele und die zukünftigen Pläne im Rahmen seiner Forschungsprogramme. Ergänzt wird dies durch Erläuterungen zur Struktur, zu Aufgaben und Visionen des Instituts. Ferner soll der Bericht dazu beitragen, die Ergebnisse unserer Arbeit, die uns bewegenden dringlichen Fragestellungen und auch die Faszination viro-logischer Grundlagenforschung der Öffentlichkeit zu vermitteln.

Wir sind zuversichtlich, dass die in diesem Bericht dokumentierte Leistungsbilanz der vergangenen zwei Jahre unsere Zuwendungsgeber, die Mitglieder des Kura-toriums und des Wissenschaftlichen Beirats sowie die Freunde und Förderer des HPI von der wissenschaftlichen und gesellschaftlichen Bedeutung unseres Instituts und unserer Forschungsarbeiten überzeugen wird.

Hamburg, im April 2009

Prof. Dr. Thomas Dobner Vorsitzender des Kollegiums

Allgemeiner Überblick

Status- und Funktionsbezeichnungen, die in diesem Dokument in der männlichen oder weiblichen Sprachform verwendet werden, schließen die jeweils andere Sprachform ein.

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Allgemeiner Überblick

Die Struktur des Heinrich-Pette-InstitutsDas Institut ist als Stiftung bürgerlichen Rechts eine gemeinnützige und selbst-ständige Forschungseinrichtung und seit 1995 selbstständiges Mitglied der Leibniz-Gemeinschaft (WGL), einem Verbund von derzeit 86 außeruniversi- tären Forschungsinstituten, die sich durch ihre über regionale und gesellschafts-relevante Bedeutung in Deutschland auszeichnen.

Das Institut wird anteilig finanziert durch das Bundesministerium für Gesundheit (BMG) und die gemeinsame Forschungsförderung der Länder, vertreten durch die Behörde für Wissenschaft und Forschung (BWF) der Freien und Hansestadt Hamburg. Ein nicht unerheblicher Anteil des Forschungsetats wird zudem im wettbewerblichen Verfahren eingeworben. Die Gelder stammen aus staatlichen Forschungsprogrammen, privaten Stiftungen und der Industrie.

Das HPI hat vier permanente wissenschaftliche Abteilungen (Molekulare Viro-logie, Allgemeine Virologie, Zellbiologie und Virologie, Tumorvirologie), zwei Forschungsgruppen (Elektronenmikroskopie und Mikrotechnologie, Moleku- lare Pathologie) sowie derzeit zwei unabhängige Nachwuchsgruppen (Zelluläre Virus abwehr, Virus-Pathogenese). Eine dritte Nachwuchsgruppe (Influenza-Patho genese) wurde Anfang 2009 besetzt und wird im Mai 2009 ihre Arbeit aufnehmen. Daneben gibt es innerhalb der Abteilungen eine Vielzahl von Arbeitsgruppen, die sich durch Drittmittel weitgehend selbst finanzieren. Die Forschungsabteilungen stellen die strukturell-thematischen Basiseinheiten dar, die in Bezug auf bestimmte humanpathogene Viren und ein spezifisches Methodenspektrum jeweils unterschiedliche Schwerpunkte setzen und damit eine sowohl thematische als auch technologische Kernfunktion innehaben.

Die Mischung permanenter und zeitlich begrenzter Organisationseinheiten gewährleistet eine breite Themen- und Technologiebasis als Kernstruktur des Instituts, die mit Forschungstätigkeiten zu aktuellen Fragen ergänzt wird. Hierdurch erreicht das HPI ein hohes Maß an Flexibilität, ohne die Kontinuität seines Forschungsprofils zu vernachlässigen. Diese Flexibilität begünstigt das rasche Aufgreifen neuer Entwicklungen, was gerade im Bereich der virusasso-ziierten Infektionskrankheiten wegen des häufigen Auftretens neuer oder ver-änderter Erreger von Bedeutung ist. Die Einteilung in wissenschaftliche Abteilungen und Gruppen wird durch zwei abteilungsübergreifende Pro-grammbereiche („Virus-Wirts-Wechselwirkungen“ und „Zelluläre Dysregu-lation“) zu einer flexiblen Matrix zusammengefasst, die den interdisziplinären Austausch und die Kooperationen innerhalb des Instituts maßgeblich prägt. Prof. Joachim Hauber, Abteilung „Zellbiologie und Virologie“, ist für den Pro-grammbereich „Virus-Wirts-Wechselwirkung“ verantwortlich und Dr. Carol Stocking, Forschungsgruppe „Molekulare Pathologie“, für den Programmbereich „Zelluläre Dysregulation“.

2

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3Gemäß seiner Satzung wird das HPI durch einen Vorstand geleitet, der zum einen aus einem Abteilungs- oder Forschungsgruppenleiter besteht, welcher als Wissen-schaftlicher Direktor alle fünf Jahre neu gewählt wird, und dem zum anderen der Leiter der kaufmännischen Abteilung als ein permanentes Mitglied angehört. Die jeweiligen Vertreter komplettieren den Vorstand. Im Berichtszeitraum ist Herr Dr. Heinrich Hohenberg wissenschaftlicher Direktor, sein Stellvertreter ist Herr Prof. Thomas Dobner. Von der kaufmännischen Seite gehört dem Vorstand als Leiter der Abteilung Herr Dr. Volker Uhl an, stellvertreten durch Herrn Jörg Schinkel.

Der Vorstand legt gegenüber dem Kuratorium Rechenschaft ab, der Wissen-schaftliche Beirat sorgt für die externe Beratung, das Kollegium für die interne wissenschaftliche Koordination. Das Kuratorium führt unter dem Vorsitz des Kurators die Aufsicht über das Direktorium und überwacht die Geschäfte der Stiftung im Hinblick auf Rechtmäßigkeit, Zweckmäßigkeit und Wirtschaftlichkeit. Es berät das Direktorium in Fragen der Forschungsziele und Struktur-angelegenheiten sowie mittelfristig in der Finanz- und Wirtschaftsplanung und der Bestellung der Mitglieder des Wissenschaftlichen Beirats. Ein externer Wissenschaftlicher Beirat evaluiert regelmäßig die Leistungen des HPI und berät in Fragen der wissenschaftlichen Ausrichtung bzw. der Programmbudgets. Als internes Beratungsgremium fungiert das Kollegium, welches in einem Turnus von etwa zwei Monaten tagt. Es ist verantwortlich für die wissenschaftliche Entwicklung des Instituts, unterstützt bei der Erstellung der Forschungsberichte und wirkt bei der Berufung von Abteilungs-, Forschungs- und Nachwuchs-gruppenleitern mit. Derzeitiger Leiter des Kollegiums ist Prof. Thomas Dobner, sein Stellvertreter ist Herr Dr. Michael Schindler.

Allgemeiner Überblick

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Allgemeiner Überblick

4Modernisierung des HPI erfolgreich abgeschlossenIm vorliegenden Berichtszeitraum konnte das Heinrich-Pette-Institut eine lange Phase reger Bautätigkeiten und Modernisierungen erfolgreich abschließen. Zu seinem 60-jährigen Jubiläum im Jahr 2008 präsentierte sich das Institut mit modernen, gut ausgestatteten und lichtdurchfluteten Labor- und Büroflächen. Auch das äußere Erscheinungsbild entspricht jetzt modernen ästhetischen und architektonischen Standards. Der neue Ersatz- und Erweiterungsbau war bereits im September 2006 eingeweiht worden. Während der knapp zweijährigen Bau-phase konnten auf Grund der günstigen Baukonjunktur deutliche Einsparungen erzielt werden, so dass der für den Neubau vorgesehene Investitionsrahmen von 16,15 Mio. Euro nicht ausgeschöpft werden musste. Daher bewilligten die Zu- wendungsgeber, das Bundesministerium für Gesundheit und der Senat der Freien und Hansestadt Hamburg, abschließend nochmals 1,8 Mio. Euro für die Sanierung des alten Seuchenlaborgebäudes des Heinrich-Pette-Instituts. So konnte der drin-gend notwendige Modernisierungsprozess vollendet werden.

Im Inneren des alten Seuchenlaborgebäudes entstanden auf 460 qm modernste Laborflächen, die neuesten L2-Sicherheitsstandards entsprechen. Außerdem wur-den die Dachfläche und die Fassade des Gebäudes aus den 60er Jahren saniert und dem Erscheinungsbild der neuen Institutsteile angepasst. Die Wärme-dämmung der Außenflächen zur effizienteren Energieeinsparung war dem Institut ein besonderes Anliegen. Der damalige Wissenschaftssenator Jörg Dräger bestätigte im Jahr 2007: „Mit der Sanierung des alten Gebäudes wird der Erneuerungsprozess der Gebäude des Heinrich-Pette-Instituts ab ge schlos sen. Damit verfügt diese exzellente Forschungseinrichtung dann über eine moderne, erstklassige For schungsinfrastruktur.“

Das renovierte Seuchenlaborgebäude mit neu gestalteter Fassade

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Unser herzlicher Dank gilt unseren Zuwendungsgebern, die die Modernisierung des Heinrich-Pette-Instituts auch in wirtschaftlich schwierigen Zeiten unterstützt und begleitet haben. Diese vertrauensvolle Unterstützung bestärkt uns darin, auch in Zukunft das hohe wissenschaftliche Ansehen des Heinrich-Pette-Instituts weiter auszubauen und die Infektionsforschung in der Metropolregion Hamburg entscheidend mitzuprägen.

Wir danken auch der Fördergemeinschaft Kinderkrebs-Zentrum Hamburg e.V., die sich im Rahmen einer Public-Privat-Partnership an den Kosten des Neubaus beteiligt hatte, und dort seit 2006 in 540 qm Laborfläche eingezogen ist. Seit zwei Jahren hat sich mit dem Forschungsinstitut Kinderkrebs-Zentrum eine äußerst erfolgreiche Partnerschaft im wissenschaftlichen, organisatorischen und adminis-trativen Bereich entwickelt: zwei Forschungskooperationen entstanden, Schlüssel-technologien werden gemeinsam genutzt und öffentlichkeitswirksame Veranstal-tungen, wie die Hamburger „Nacht des Wissens“, gemeinsam durchgeführt.

60 Jahre HPIIm Jahr 2008 konnte das Heinrich-Pette-Insitut auf sechzig erfolgreiche Jahre seiner Instituts-geschichte zurückblicken und feierte dieses Jubi-läum im Rahmen einer exzellenten Pette-Lecture mit Herrn Prof. Rudolf Jaenisch und einem großen Sommerfest für alle Mitarbeiterinnen und Mitarbeiter, Freunde des Instituts und deren Familien. Das HPI ehrte Prof. Rudolf Jaenisch, Gründungsmitglied des „Whitehead“ Instituts in Cambridge, USA, für seine herausragenden Arbeiten im Bereich des therapeutischen Klo-nens, transgener Mausmodelle und der Stamm-zellforschung mit der Vergabe der Heinrich-Pette-Lecture 2008.

Prof. Rudolf Jaenisch (1975 bis 1985 auch Abteilungsleiter am HPI) war nach Prof. Peter K. Vogt (2006) und Prof. Thomas Shenk (2007) der dritte Ehrengast der seit 2006 jährlich stattfindenden Heinrich-Pette-Lecture.

Allgemeiner Überblick

5

Prof. Rudolf Jaenisch hält die Pette-Lecture 2008

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6Im Jubiläumssommer 2008 besuchte auch die Hamburger Wissenschafts-senatorin Frau Dr. Herlind Gunde-lach das Heinrich-Pette-Institut, um sich vor Ort umfassend über die Forschungsschwerpunkte, die Labore, Technologien, Strukturen und Visionen des Traditionsinstituts zu informieren. Begleitet wurde sie von Staatsrat Bernd Reinert und Mit arbeitern der Behörde für Wissen schaft und Forschung. Die Besucher gruppe zeigte sich beein-druckt von der Neustrukturierung des Instituts mit hochmodernen Technologie- und Methodenplatt-formen und neuen attraktiven Arbeitsbedingungen für Nach-wuchsforscher. Ein wichtiger Aspekt war die intensivierte Vernetzung wissen-schaftlicher Arbeiten nicht nur innerhalb des HPI, sondern vor allem mit regio-nalen, nationalen und internationalen Part nern. Die Senatorin: „Forschung funktioniert heute nur im Ver bund, und ich sehe, dass Sie hier am HPI exzellente Netzwerke knüpfen und national und international ein starker Partner in inno-vativen Forschungsverbün den sind.“

Offen für den DialogDas Heinrich-Pette-Institut stellt seine Forschung im Dialog mit der Öffentlichkeit transparent dar und hat seine Presse- und Öffentlichkeitsarbeit seit 2007 weiter ausgebaut. Insgesamt berichteten nationale und internationale Printmedien im Berichtszeitraum 214mal über Forschungsergebnisse am HPI, Online-Me dien 735mal und 22mal wurden wissenschaftliche Highlights des HPI in Rundfunk und Fernsehn porträtiert. Insbesondere die Pressemeldung zur „Molekularen Schere gegen HIV-1“ fand weltweit große Beachtung.

Darüber hinaus öffnet das HPI seine Türen für interessierte Mitbürger. So besuchten bei der 2. Hamburger „Nacht des Wissens“ im Juni 2007 wieder mehr als 900 interessierte Gäste, Familien, Schülerinnen und Schüler das Institut und waren

Besuch der Wissenschaftssenatorin Dr. Herlind Gundelach am HPI (von li nach re Dr. H. Hohenberg, Senatorin Dr. H. Gundelach, Staatsrat B. Reinert)

Allgemeiner Überblick

Bericht zum 60-jährigen Jubiläum des HPI im NDR-Fernsehn

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7begeistert bei vielen Mitmachangeboten und Präsentationen unter dem Titel „Faszination Virusforschung“ dabei. Als einen der Gäste konnte das HPI gemein-sam mit dem Forschungsinstitut Kinderkrebs-Zentrum auch den damaligen Wissenschaftssenator Jörg Dräger begrüßen.

Bereits zweimal war der Hamburger Zweig der international tätigen Initiative „Common Purpose“ bei Prof. Joachim Hauber am HPI zu Gast. Führungskräfte Hamburger Firmen sowie aus Behörden und gemeinnützigen Organisationen kamen im Rahmen eines mehrmonatigen Matrix-Programmes, um sich über aktuelle Forschungsprojekte im Bereich HIV/AIDS und deren gesellschaftliche Bedeutung zu informieren.

Auch naturwissenschaftlich interessierte junge Talente konnten bei zwei weiteren Veranstaltungen gezielt angesprochen werden: Das HPI beteiligte sich im September 2007 an einem von 1100 Schülern besuchten Schülerkongress zu den Themen Molekularbiologie, Biomedizin und Biotechnologie in Hamburg. Im Sommer 2008 konnten dann Schülerinnen, Schüler und Studenten auf Einladung des Heinrich-Pette-Instituts an einem Symposium zur Stammzellforschung teil-nehmen. Sie lernten aktuelle Forschungsarbeiten dreier internationaler Wissen-schaftler zur Leukämieforschung, Immuntherapie gegen Krebs und zu modernen Bildgebenden Verfahren in der Stammzellforschung kennen. Der Schüler- und Studententag fand im Rahmen des internationalen Wilsede-Meetings „Modern Trends in Human Leukemia“ statt (mitorganisiert von Dr. Carol Stocking, HPI) und wurde mit einem persönlichen Gespräch der jungen Menschen mit Prof. Sir Timothy Hunt, Nobelpreisträger der Medizin 2001, abgeschlossen.

Gäste bei der „Nacht des Wissens“ am Elektronenmikroskop

Allgemeiner Überblick

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8 Preisverleihungen und EhrungenDas HPI-Forschungsprojekt „Eradikation proviraler HIV-1 DNA aus Patienten-zellen“ war beim GO-Bio Wettbewerb 2007 des Bundesministeriums für Bildung und Forschung (BMBF) erfolgreich. Das Projekt unter der Leitung von Prof. Joachim Hauber gehört zu den sieben Siegern, die im Juli 2007 bei der zweiten Förderrunde des Wettbewerbs aus 85 Mitbewerbern nominiert wurden. Für die nächsten drei Jahre stehen den Forscherteams insgesamt 20 Millionen Euro zur Verfügung, um eine wissen-schaftliche Idee zu einem markt-fähigen Produkt entwickeln zu können; davon gehen 2,2 Mio. Euro an das Projekt von Prof. Hauber und seine Kooperations-partner. Die Antragstellung des HPI profitierte insbesondere von einer guten und engen Zu - sammenarbeit mit der Patent-Manage ment-Agentur Ascenion. Ascenion hat sich klar auf das Gebiet der „Life-Sciences“ fokus-siert und betreut das HPI in allen Aspekten der Patentierung und Kommerzialisierung ihrer For-schungsergebnisse.

Prof. Joachim Hauber erhielt 2008 die Ehre, in den wissenschaftlichen Beirat der Robert-Koch-Stiftung berufen zu werden, der u.a. unter Schirmherrschaft des Bundespräsidenten jährlich den renommierten Robert-Koch-Preis verleiht. Außerdem wurde er für die nächsten fünf Jahre in den Universitätsrat der „Veterinärmedizinischen Universität Wien“ berufen.

Prof. Wolfgang Deppert wurde damit ausgezeichnet, die „Centers of Excellence Lecture“ 2007 an der Keio University Tokio mit dem Titel „Mutant p53 Gain of Function: in vivo Phenotype and in vitro Molecular Analyses“ halten zu können. Er erhielt diese Ehrung für seine grundlegenden Studien zur Funktion des Tumorsuppressors P53 und dessen mutierte Varianten bei der Kontrolle des Zellzyklus und der Tumorgenese sowie für die Entwicklung eines transgenen Mausmodells zur Mammakarzinogenese.

Preisverleihung zum GoBio Wettbewerb 2007 durch Ministerialrat E. Warmuth (BMBF, im Bild li) an Prof. J. Hauber (im Bild re) bei der Biotechnica 2007

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9Dr. Heike Helmbold wurde im Juli 2007 mit dem Promotions-Preis des Freundes- und Fördervereins Chemie der Universität Hamburg ausgezeichnet. Sie erhielt den Preis für ihre Studien zur Dysregulation der G1-Phase, die sie im Rahmen ihrer Promotionsarbeit in der Abteilung „Tumorvirologie“ erfolgreich abschließen konn-te. Die Dys regu lation der G1-Phase ist häufig der Einstieg in die Tumor entstehung.

Fast immer sind dabei der p16INK4A-pRb und/oder der ARF-p53 Signal-weg ge stört, meistens durch Mutation oder Dele tion der Tumor -suppressor gene p16IN-K4A, ARF und p53. Die Reak ti vierung der Signal-wege durch Rekonsti-tution der inaktivierten Prote ine könnte eine Mög lich keit sein, die Kon trolle über die ent-gleiste Prolife ration wie-der herzustellen.

Im Jahr 2007 wurde erstmals der neu ausgelobte Nachwuchspreis des Heinrich-Pette-Instituts verliehen. Der Preis soll zukünftig aktuelle hochrangige Publika-tionen von Diplom- bzw. Promotionsarbeiten des Instituts fördern. Ausgezeichnet wurden für die besten Publikationen im Jahr 2006 Barbara Fries für ihre Arbeit „Analysis of Nucleocytoplasmic Trafficking of the HuR Ligand APRIL and its Influence on CD83 Expression“ im Journal of Biological Chemistry und Dr. Heike Helmbold für ihre Publikation „Regulation of cellular senescence by Rb2/p130“ in Oncogene. Wir beglückwünschen beide für ihre hervorragenden Publikatio nen und je 500 Euro Preisgeld. Das Auswahlverfahren für 2008 ist derzeit noch nicht abge-schlossen.

Dr. Gabriela B. Iwanski gewann den Jürgen Lüthje-Promotionspreis 2007. Die Doktorandin von Dr. Carol Stocking und Prof. Klaus Harbers hatte über „Unter-suchungen zur Rolle des Transkriptionsfaktors C/EBPalpha bei der normalen und aberranten Hämatopoese mit Hilfe der RNA-Interferenz Methode“ gearbeitet und diese Arbeiten im März 2007 mit der Promotion erfolgreich abgeschlossen. Der vom Freundes- und Förderkreis des Universitätsklinikums verliehene und mit 1250 Euro dotierte Preis wurde am 14. Dezember 2007 überreicht.

Dr. Afra Engelmann (Forschungsgruppe „Molekulare Pathologie“) wurde 2007 mit dem Mildred-Scheel-Stipendium der Mildred-Scheel-Stiftung für Krebs forschung ausgezeichnet.

Prof. Vladimir Prassolow, Gastwissenschaftler der Forschungsgruppe „Molekulare Pathologie“, gewann 2007 den EMBO Journal-Cover Contest für das beste Titel foto des EMBO-Journals.

Verleihung des Promotions-Preises durch Prof. Heck (UKE) an Dr. Heike Helmbold

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10Personelle Veränderungen in den Jahren 2007 und 2008Nach einer Phase begrenzter und beengter Büro- und Laborflächen in den Vor-jahren, bedingt durch umfassende Baumaßnahmen, konnten die Abteilungen und Forschungs- sowie Nachwuchsgruppen am Heinrich-Pette-Institut ab 2007 wieder vollständig besetzt werden. Insgesamt stehen dem HPI nun 30 Prozent mehr hochmoderne Laborflächen zur Verfügung.

Adenovirusforschung am HPI etabliert

Mit Herrn Prof. Thomas Dobner gelang es dem HPI 2006 einen international anerkannten Experten für Adenoviren zu berufen. So konnte Prof. Dobner die Ende 2006 neu eingerichtete Abteilung „Molekulare Virologie“ mit dem For-schungsschwerpunkt humaner Adenoviren im Berichtzeitraum erfolgreich eta-blieren. Humane Adenoviren sind für meist harmlos verlaufende Infektionen der oberen Atemwege und der Bindehaut des Auges verantwortlich. In Nagern kön-nen Adenoviren jedoch Tumore erzeugen. Seit Jahrzehnten dienen Adenoviren insbesondere als Modell für grundlegende Fragestellungen zur Funktion viraler Kontrollproteine in der Steuerung des produktiven Replikationszyklus und in der Virus-vermittelten Zelltransformation. In ihrer Veröffentlichung im Fachjournal Journal of Virology beschrieb die Abteilung erstmals eine Beteiligung des adeno-viralen E1B-156R Proteins bei der Entartung von Nagetierzellen (J. Virol. 2007, 81: 95-105).

Ein weiterer Schwerpunkt der Abteilung liegt auf Untersuchungen der virusregu-lierten Vorgänge des Kerntransports und Proteinabbaus sowie auf Analysen zur Rolle der PML-NB Zellkern-domänen (PML-NBs) in der vira-len und zellulären Proliferations-kontrolle. Prof. Thomas Dobner und Dr. Barbara Härtl zeigten erstmals, dass ein Pro tein kom-plex, der normalerweise Brüche im Erbgut repariert und wichtige Mechanismen in Zellen steuert, während der Tumorbil dung durch Adenoviren manipu liert wird (Oncogene, 2008, 27: 3673-3684). Mre11, ein Teil dieses Reparaturkomplexes, ist zentra les Ziel der Adenoviren.

Prof. Dobner ist als Mitglied der Fakultät für Mathematik, Informatik und Natur-wissenschaften (MIN) seit 2007 ebenfalls im neuen Bachelor-/Masterstudiengang der Universität Hamburg eingebunden. Die ersten Bachelorstudenten konnten am HPI im Rahmen des Vertiefungsmoduls „Methoden der molekularen Virolo gie und Zellbiologie“ bereits ihre Fallstudien und Abschlussarbeiten fertigstellen.

Verteilung von Mre11 (grün) und E1B-55K (rot); Wild typ (links) und Mutante (rechts)

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11Nachwuchsgruppe „Virus-Pathogenese“

Ende 2007 konnte eine zweite Nachwuchsgruppe mit dem Arbeitsschwerpunkt „Virus-Pathogenese“ am Heinrich-Pette-Institut starten. Das HPI freut sich hierfür Dr. Michael Schindler, einen mehrfach ausgezeichneten Jung forscher aus Ulm, an das Institut geholt zu haben. Ermöglicht wurde dies durch Fördergelder aus dem Wettbewerbs verfahren „Pakt für Forschung und Innovation“. Dr. Michael Schindler beschäftigt sich seit mehreren Jah ren mit der Frage,

warum HI-Viren beim Menschen AIDS hervor - rufen, während Infektionen mit nahe verwandten SI-Viren bei Affen meist harmlos verlaufen. Bereits 2007 erhielt er den renommierten Paul-Ehrlich- und Ludwig-Darmstaedter Nachwuchspreis, gefolgt vom Deutschen AIDS-Preis für seine Erkenntnis, dass das virale Nef-Protein hierfür verantwortlich sein könnte. Neben der Rolle des Virusproteins Nef unter- sucht die Nachwuchsgruppe auch die Funktion der akzessorischen viralen Proteine Vpu und Vpr. Da das pathogene Potenzial eines Virus nicht nur von viralen, sondern auch von zellulären Faktoren ab- hängt, und die Ent stehung von AIDS eine Summe

diverser Wechselwirkungen ist, versucht die Forschungsgruppe zudem grund-legende Unterschiede im Immunsystem infizierter Wirte zu identifizieren, die den Krankheitsverlauf beeinflussen.

Wissenschaftliche Koordination und Öffentlichkeitsarbeit

Im Jahr 2007 konnte das HPI eine Stelle für einen Wissenschaftlichen Koordinator/Vorstandsreferenten schaffen. Sie wurde im April 2007 mit Dr. Jörg Maxton-Küchenmeister besetzt, der zum Januar 2008 als Generalsekretär an die Ham- burger Akademie der Wissenschaften wechselte. Im Hinblick auf zunehmende Aufgaben im Bereich der Öffentlichkeitsarbeit und wissenschaftskommunika-tiven Aktivitäten hat das HPI die Vorstandsreferentenstelle im Jahr 2008 halbiert und so zusätzlich eine halbe Stelle für Presse- und Öffentlichkeitsarbeit ein-gerichtet. Die verbliebene halbe Vorstandsreferentenstelle und eine halbe LCI-Koordinatorenstelle, deren Finanzierung die Stadt Hamburg zunächst für zwei Jahre übernahm, hat seit 2008 Frau Dr. Nicole Nolting in Personalunion inne. Frau Dr. Nolting war vorher als wissenschaftliche Mitarbeiterin an den Uni-versitäten Bochum und Göttingen beschäftigt und hat dort u. a. an der Entwick-lung der Graduate School-Konzepte mitgewirkt und die Bewerbung innerhalb der Exzellenzinitiativen unterstützt. Als Referentin für Presse- und Öffentlich-keitsarbeit konnte das HPI Frau Dr. Angela Homfeld gewinnen, die diesen Bereich am Institut bereits seit 2004 als freie wissenschaftliche Mitarbeiterin betreut hatte. Das Institut strebt eine Wiederaufstockung der Vorstandsreferentenstelle auf eine volle Stelle an, ohne die Stellen für Öffentlichkeitsarbeit/wissenschaftskom-munikative Aktivitäten und für die LCI-Koordination zu beschneiden.

Dr. Michael Schindler

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12Wissenschaftliche HighlightsGrundstein für neuartige HIV-Therapie

Das Highlight des Jahres 2007 war zweifellos die hervorragende Arbeit von Dr. Ilona Hauber und Prof. Joachim Hauber sowie ihren Kooperationspartnern zur „Eradikation proviraler HIV-1 DNA aus Patientenzellen“ mittels einer maßge-schneiderten Rekombinase. Die in Science (Sarkar et al., 2007, 316: 1912-1915) veröffentlichte Studie ermöglicht erstmals die quantitative Entfernung chromo-somal-integrierter Proviren aus HIV-infizierten Zellen und bietet somit ein neu-artiges biotechnologisches Verfahren in der HIV-Therapie. Besondere Beachtung fand die Publikation auch in Form eines von Alan Engelmann geschriebenen zweiseitigen Kommentars in Science.

Das Ausgangsmaterial für die neue Methode war eine natürlich vorkommende Cre-Rekombinase des Bakteriophagen P1. Sie katalysiert spezifische symme-trische Sequenzen von 34 bp Länge, die dem HIV-Erbgut nur entfernt ähnlich sind. Der Kooperationspartner am Max-Planck-Institut für Molekulare Zell-biologie und Genetik in Dresden, Dr. Frank Buchholz, entwickelte daraus mit Hilfe der moleku- la ren Evolution in 126 Zyklen eine neue hochspezifische Tre-Rekombinase, die eine natürlich vorkommende asymmetrische HIV-1 LTR-Sequenz erkennt und rekombiniert. Die Gruppe am HPI zeigte, dass die genthera-peutisch eingeschleuste Tre-Rekombinase in infizierten Zell-kulturen etwa zehn Wochen be nötigt, um alle Proviren aus der Kultur zu entfernen.

Dr. Frank Buchholz, Dr. Ilona Hauber und Prof. Joachim Hauber (von li nach re)

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13

Blockade bei HIV-Infektionen entschlüsselt

Wissenschaftler des Heinrich-Pette-Instituts entdeckten eine Blockade in ruhen-den Blutzellen, die eine Vermehrung des AIDS-Erregers HIV-1 unterbindet. Der zentrale Schalter dieser Blockade ist ein zelluläres Protein, der Glucocorticoid-rezeptor (GR). Dr. Klaus Wiegers, Dr. Wolfgang Bohn und Kolle gen des HPI veröffentlichten ihre Ergebnisse in der online-Ausgabe des Fach journals Virology (Wiegers et al., 2008, 375: 73-84). Während das HI-Provirus in aktivierten Blutzellen in den Zellkern transportiert und im Erbgut eingebaut wird, bleiben die Proviren in ruhenden Blutzellen im Zytoplasma und werden nach wenigen Stunden abgebaut. Die HPI-Forscher überwanden diese frühe Blockade der HIV-Infektion in ruhenden, nicht ausgereiften Blutzellen, indem sie den Glucocorti-coid-Rezeptor aktivierten. Sie zeigten erstmals, dass Steroide die Blockade in diesen Zellen aufheben können, und vermuten, dass sich bei Stress und Steroid-therapien die Anzahl der Blutzellen erhöht, die für eine produktive HIV-Infektion empfänglich sind.

1 2

3

HIV Lebenszyklus HIV Entfernung

Therapierte Wirtszelle

Infektion

Integration

Virusvermehrung

Tre Rekombinase

Eradikation proviraler HIV-1 DNA aus infizierten Zellen (Grafik: Dr. F. Buchholz, MPI Dresden)

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14Nef schützt Affen vor T-Zell-Verlust

Während HIV-1 bei Menschen die Immunschwäche AIDS hervorruft, verlaufen Infektionen mit dem nahe verwandten SI-Virus in Mangabenaffen meist harm-los. In seinen früheren Studien zeigte Dr. Michael Schindler (Nachwuchsgruppe „Virus-Pathogenese“), dass hierfür das virale Nef-Protein mitverantwortlich ist. Beide Virusarten besitzen dessen Erbinformation, Nef-Allele aus HIV und SIV kodieren aber offensichtlich für unterschiedliche Funktionen. Im Juli 2008 be- schrieben Dr. Michael Schindler und Prof. Frank Kirchhoff (Universität Ulm) im renommierten online-Magazin PLoS Pathogens (2008;doi:10.1371/ppat.1000107), wie Nef vor einer tödlichen Immunschwäche schützt. Außerdem zeigten sie erst-mals, dass dieser Schutz tatsächlich eine Rolle spielt, wenn Mangabenaffen in freier Wildbahn infiziert werden. Nur Nef aus SIV-infizierten Tieren mit einem stabilen Immunsystem kann vor einem Verlust von T-Zellen schützen.

MEF2C moduliert Blutzellen

Veränderungen in Genen für Transkriptionsfaktoren sind maßgeblich an der Ent-stehung von akuten Leukämien beteiligt. Diese Transkriptionsfaktoren werden u.a. durch Zytokine gesteuert und regulieren im Knochenmark die Entstehung von reifen Blutzellen aus hämatopoetischen Vorläufer- oder Stammzellen. Dr. Carol Stocking und Mitarbeiter ihrer Forschungsgruppe „Molekulare Pathologie“ entdeckten, dass die Regulation des Gens für den MEF2C-Transkriptionsfaktor in akut myeloischen Leukämien des Kindesalters verändert ist. MEF2C reguliert normalerweise die Entwicklung muskulärer und kardiovaskulärer Gewebe. In Blood (2008, 111: 4532-4541) zeigte die Forschungsgruppe, dass das MEF2C-Gen auch in hämatopoetischen Stammzellen, bei der Differenzierung reifer myelo-ischer und lymphoider Blutzellen sowie in bestimmten AML-Zellen aktiviert ist. In diesen AML-Zellen steht der Transkriptionsfaktor unter der Kontrolle des leukämischen Fusionsproteins MLL-ENL.

Hepatitis B Viren und intrazelluläre Vesikel

Dr. Mouna Mhamdi, Dr. Hüseyin Sirma und Kollegen des HPI konnten massive Umstrukturierungen nachweisen, die im Verlauf einer hepadnaviralen Infektion in Hepatozyten entstehen. Dies äußert sich in der Bildung zahlreicher intrazellulärer Vesikel (VCV) unterschiedlicher Größe. Diese intra-zellulären Vesikel sind dynamische Struktu ren und unterliegen sowohl homo- als auch heterotypischer Fusion. Ein Teil der VCV leitet sich wahrscheinlich von der äußeren Kern mem bran und vom rauhen endoplasmatischen Retikulum (rER) ab. Die HPI-Forscher zeigten, dass ein Teil der VCV sowohl Virionen als auch subvirale Partikel (SVPs) enthalten. Erstmals konnte die lang diskutierte Frage beantwortet wer- den, ob die für Hepatitis B Viren typischen SVPs über denselben Morphogeneseweg formiert und warum sie im Überschuss produziert werden. Die Daten wurden in Hepa tology (2007, 46: 95-106) publiziert.

Intrazelluläre Vesikel (Bild: Dr. H. Hohenberg)

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15Transgenes Mausmodell zu Mammakarzinomen

Wissenschaftler der Abteilung „Tumorvirologie“ entwickelten ein transgenes Mausmodell, mit dem die Rolle von Punktmutationen im p53-Gen während ein-zelner Stadien der Mammakarzinogenese und der Entstehung von Metastasen untersucht werden kann. Dieses Modell, in dem in erwachsenen Mäusen durch Anschalten der Tumor-auslösenden SV40 Proteine Brusttumore induziert werden können, ermöglicht erstmals quantitative Aussagen zur Funktion von p53-Mutan-ten in einer definierten isogenen Kohorte. In ca. 30% aller Brusttumore bei Menschen finden Kliniker veränderte dysfunktionelle P53-Proteine, denen defi-nierte Punktmutationen im p53-Gen zugrunde liegen. Mit Hilfe ihres neuen Mausmodells können Prof. Wolfgang Deppert und seine Kollegen untersuchen, wie sich analoge Punktmutationen in Brustdrüsen der Maus auf den Krankheitsverlauf und die Bildung von Metastasen auswirken. Die Forscher stellten hierfür in trans-genen Mäusen p53-Mutanten unter Kontrolle eines Brustdrüsen-spezifischen Promotors und kreuzten diese Mäuse in die oben beschriebenen SV40-Mäuse ein. Erste funktionelle und histopathologische Befunde zeigten, dass p53-Mutanten bei der Progression der Mammakarzinome eine wichtige Rolle spielen und mit einem erhöhten metastatischen Potenzial der Tumorstammzellen korrelieren. Die Wissenschaftler konnten ihre Studien im International Journal of Cancer veröffent-lichen (Heinlein et al., 2008, 122: 1701-1709).

Darstellung funktionalisierter Nanosomen

Dr. Heinrich Hohenberg und Dr. Rudolf Reimer ist erstmals die Darstellung so genannter funktionalisierter aggregierter Nanopartikel im Transmissions-Elektro-nenmikroskop gelungen. In einem Gemeinschaftsprojekt unter Leitung von Prof. Horst Weller (Universität Hamburg) wurden spezifizierte superparamagnetische MnFe2O4-Nanopartikel für die Magnetresonanz-Tomograpie entwickelt. Die HPI-Forscher konnten diese mit Liposomen und Micellen aggregierten Nanosomen in ultradünnen Carbo-Cell-Mikrokapillaren elektronenmikroskopisch in ihrer Dyna-mik darstellen. Die Ergebnisse wurden im Juli 2007 in Nanoletters publiziert (Tromsdorf et al., 2007, 7: 2422-2427).

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16Hohes Drittmittelaufkommen am HPIDas hohe Drittmittelaufkommen des HPI bewegt sich in den letzten Jahren durch-schnittlich auf einem Niveau von 22% bezogen auf die Grundfinanzierung. Nach Eli minierung der Bauinvestitionen erreicht das Institut sogar einen Drittmittelanteil von fast 30%, so dass es damit einen Spitzenplatz innerhalb der Leibniz-Gemein-schaft erreicht. Drittmittelprojekte, die in den Jahren 2007 und 2008 besondere Beachtung gefunden haben, sind im Folgenden aufgelistet.

HIV-Forschung

Prof. Joachim Hauber gewann 2007 den vom BMBF geförderten GO-Bio-Wett- bewerb und konnte für die nächsten drei Jahre 2,2 Mio. Euro für das Koope-rationsprojekt „Eradikation proviraler HIV-1 DNA aus Patientenzellen“ einwer-ben. Mit Hilfe der Fördergelder soll die neu entwickelte „Molekulare Schere“, die Tre-Rekombinase, optimiert und von der Grundlagenforschung in ein anwend-bares Produkt weiter entwickelt werden. Zunächst wird die Herstellung der Tre-Rekombinase vereinfacht und optimiert. Da die Rekombinase mittels soma-tischer Gentherapie in Zellen eingebracht wird, müssen hierfür außerdem geeig-nete Vektoren gefunden und verbessert werden. Anschließend wird das Enzym im Tiermodell auf seine Verträglichkeit und Wirksamkeit hin untersucht. Ziel ist es, eine Methode zu entwickeln, durch die hämatopoetische Stammzellen aus AIDS-Patienten isoliert, mit dem Rekombinasegen-Vektor behandelt und anschließend wieder in den Blutkreislauf der Patienten zurückgeführt werden können.

Prof. Joachim Hauber ist neben Prof. Tim Brümmendorf Koor-dinator des Verbundvorhabens „Combating Drug Resistance in Chronic Myeloid Leukemia and HIV-1 Infection“, für das im BMBF-Förderverfahren „Inno- vative Therapieverfahren auf molekularer und zellulärer Basis“ für die nächsten drei Jahre eben-falls 2,1 Mio. Euro bewilligt wurden. Teilnehmer an diesem Forschungsverbund sind neben dem HPI Arbeitsgruppen des Universitätsklinikums Hamburg-Eppendorf und der Universitäten Hamburg und Lübeck.

Ein kürzlich bewilligter DFG-Forschungsantrag der Nachwuchsgruppe „Virus-Pathogenese“ unter Leitung von Dr. Michael Schindler mit einem Umfang von etwa 300T Euro soll klären, wie HIV-1 im Gegensatz zu nahe verwandten SI-Viren und HIV-2 die Eisenaufnahme infizierter Zellen verstärkt.

Projektgruppe „Combating Drug Resistance“

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17Mechanismen der Onkogenese

Dr. Carol Stocking, Leiterin der Forschungsgruppe „Molekulare Pathologie“ am HPI, erhält in einer neuen Förderrunde 0,3 Mio. Euro von der José Carreras Stiftung. Seit der ersten Förderrunde im Jahr 2002 würdigt und unterstützt die Stiftung ihre Forschungsarbeit mit annähernd 1 Mio. Euro. Inhalt des aktuellen Projekts ist die Bedeutung der MEF2C und MEF2D Transkriptionsfaktoren bei der Entstehung der akuten Leukämie.

Eine der häufigsten molekularen Veränderungen in der Akuten Myeloischen Leukämie (AML) ist die FLT3-Mutation. Frau Dr. Stocking und ihre Kollegen der Forschungsgruppe „Molekulare Pathologie“ sind Teil eines nun verlängerten Ver bundprojektes, in dem Onkogen-Netzwerke und FLT3-abhängige Signalwege in der AML entschlüsselt werden sollen. Das Verbundprojekt wird bis 2011 mit insgesamt 2,76 Mio. Euro gefördert.

Die Erich und Gertrud Roggenbuck-Stiftung zur Förderung der Krebsforschung und die Wilhelm-Sander-Stiftung unterstützen die Abteilung „Molekulare Viro-logie“ des HPI mit jeweils einer Promotionsstelle zuzüglich Sachmitteln zum Studium molekularer Mechanismen der viralen Onkogenese.

Die „German Israeli Foundation“ fördert ein Projekt der Abteilung „Tumor virologie“ unter Leitung von Prof. Wolfgang Deppert zu weiteren Studien von mutantem p53; der For- schungsverbund der Krebshilfe för-dert Arbeiten zur „Beteiligung von Tumorstamm-/Progenitorzellen am Aufbau ihrer zellulären Umge bung und der metastatischen Nische“. TIME (Disseminated Tumor Cells as Targets for Inhibiting Metastasis of Epithelial Tumors) heißt ein neues vom Verein zur Förderung der Krebs-forschung e.V. gefördertes Verbund-

projekt unter Leitung des Universitätsklinikums Hamburg-Eppendorf (UKE). Prof. Wolfgang Deppert erhält als einer der Partner über drei Jahre eine zusätz-liche Wissen schaftlerstelle sowie Sachmittel.

Nanotechnologie in der Tumordiagnostik

„TOMCAT“ ist ein interdisziplinäres Konsortium aus sechs Hamburger und Schleswig-Holsteiner Forschungsinstituten und einem Industriepartner (Philips Forschungslaboratorien, Hamburg), an dem die Forschungsgruppe „Elektronen-mikroskopie und Mikrotechnologie“ unter Leitung von Dr. Heinrich Hohenberg beteiligt ist. Es hat sich zum Ziel gesetzt, unter Einsatz neuester nanotechnolo-gischer Verfahren in der Magnetresonanztomographie, hochspezifische, zielge-

TIME-Projektgruppe

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18richtete Kontrastmittel zu entwickeln, die auf molekularer Ebene im Millimeter-maßstab kleinste Gewebeveränderungen oder Metastasen erkennen können. Die Wissenschaftler verwenden hierfür so genannte SPIOs. Das sind superparamag-netische Eisenoxid-Partikel, die nur wenige Nanometer groß sind und an spezi-fische Tumormarker gekoppelt werden können. Im Rahmen des Projektes unter-sucht die HPI-Forschungsgruppe, wie diese Nanopartikel an Zellen und Gewebe binden, wie sie abgebaut oder eingelagert werden. So soll neben der makrosko-pischen Sichtbarmachung der Tumoren mittels MRT auch die Wirkungsweise der Kontrastmittel im Mikro- und Nanokosmos auf zellulärer und molekularer Ebene analysiert werden. Das Bundes minis terium für Bildung und For schung (BMBF) fördert „TOMCAT“ mit 2,8 Mio. Euro, von denen allein 400T Euro ans HPI zur Forschungs gruppe von Herrn Dr. Hohenberg fließen.

Bildgebende Verfahren am HPI

Im Rahmen des Ausbaus der strukturanalytischen Forschung im Bereich Infek-tionskrankheiten am Standort Hamburg erhält das HPI ein tomographisches Cryo-Elektronenmikroskop (CTEM), mit dem Krankheitserreger in intakten Zellen und Geweben lebensnah und räumlich dargestellt werden können. Das rund 800T Euro teure Großgerät soll 2009 in Betrieb gehen. Dr. Heinrich Hohen-berg konnte das Gerät im Rahmen des „Paktes für Forschung und Innovation“ erfolgreich einwerben.

Antikörper-gekoppelte SPIOs für die Krebsdiagnostik (Grafik: Roche, modifiziert durch R. Ittrich, UKE)

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19HPI fördert wissenschaftlichen NachwuchsDie Ausbildung wissenschaftlichen Nachwuchses nimmt am HPI traditionsge-mäß einen wichtigen Platz ein. Der Schwerpunkt liegt dabei bei der Qualifizierung Postgraduierter. Seit 2007 hat sich Prof. Thomas Dobner darüber hinaus erfolg-reich am Bachelor-/Masterprogramm der MIN-Fakultät der Universität Hamburg eingebracht und bietet innerhalb des Lehrplans Module zur Molekularen Viro-logie an. Mit einer deutlich gestiegenen Betreuung von Praktikanten (39 in den Jahren 2007 und 2008) kommt das Institut dem gestiegenen Interesse junger Menschen entgegen, die vor ihrer Entscheidung für eine naturwissenschaftliche Ausbildung Einblick in den Laboralltag nehmen möchten.

Neue Aus- und Weiterbildungskonzepte werden im Folgenden erläutert:

Mobilitätsprogramm

Im Jahr 2007 hat der Vorstand des HPI ein Mobilitätsprogramm für Dokto-randen initiiert. Im Rahmen dieses Programms gewährt das HPI Stipendien von 5000 Euro, um Doktoranden einen Forschungsaufenthalt in einem auslän dischen Labor zu ermöglichen. Ein weiteres Ziel des Mobilitätsprogramms ist es, jungen Forschern aus dem Ausland die Möglichkeit zu geben, für begrenzte Zeit am HPI zu arbeiten.

Zwei junge Wissenschaftlerinnen des HPI wurden bereits im Rahmen dieses Programms gefördert, um in ausländischen Forschungseinrichtungen neueste Techniken und Methoden zu erlernen: Birte Niebuhr für einen Aufenthalt in Los Angeles (Arbeiten zur Nutzung der SNP-ChIP-Technologie zur Analyse von Gen-veränderungen während der Tumorgenese) und Sabrina Schreiner für einen Aufenthalt in Montreal (genetische und biochemische Untersuchungen eines adenoviralen Ubiquitinligase-Komplexes). Zusätzlich erhielt Alession Medeyski, Doktorand an der Universität Neapel, Unterstützung für einen Aufenthalt am HPI bei Dr. Wolfgang Bohn.

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HPI-Nachwuchsgruppen

Mit der Einrichtung von Nachwuchsgruppen initiierte das HPI ein Programm zur Förderung von Nachwuchswissenschaftlern, das den Leitern dieser For-schungsgruppen zu einem frühen Zeitpunkt der wissenschaftlichen Karriere die Möglichkeit bietet, in höchstem Grad selbstständig zu arbeiten. Zudem versetzt es das Institut zusätzlich in die Lage, flexibel und schnell auf neue strategisch-wissenschaftliche Anforderungen einzugehen sowie neueste For schungsmethoden und Techniken zu etablieren.

Neben Dr. Adam Grundhoff, der die Nachwuchsgruppe „Zelluläre Virusabwehr“ führt, ist es dem Vorstand des HPI 2007 gelungen, Dr. Michael Schindler vom Insti tut für Virologie des Uni versitätsklinikums Ulm als Nachwuchsgruppenleiter für die neue Arbeitsgruppe „Virus-Pathogenese“ des HPI zu gewinnen. Anfang des Jahres 2009 konnte auch die dritte Nachwuchsgruppe mit Dr. Gülsah Gabriel, derzeit an der Universität Marburg (mit Gastaufenthalt an der Uni versität Oxford), erfolgreich besetzt werden. Frau Dr. Gabriel wird im Mai 2009 ihre Arbeit beginnen und trägt dazu bei, das virologische Forschungsfeld des HPI mit dem Thema Influenzaviren zu erweitern. Ihre wissenschaftlichen Arbeiten bele-gen ein hohes Maß an wissenschaftlichem Potenzial, und das HPI ist glücklich, die dritte Nachwuchsgruppe nun unter die Leitung einer Wissenschaftlerin stellen zu können.

LCI Graduate School „Modelsystems of Global Infectious Diseases”

Das HPI ist über seine Betei-ligung am Forschungsver-bund Leibniz Center for Infection (LCI) auch in der LCI Graduate School enga-giert und leistet damit einen wesentlichen Beitrag zu einer hochqualifizierten Ausbil dung wissenschaftlichen Nach - wuchses. Die interdisziplinäre Graduiertenausbildung startet im Mai 2009. Das Programm vernetzt die führenden Insti-tute im Bereich der Infektionsforschung im Raum Hamburg: die drei Leibniz-Institute Bernhard-Nocht-Institut für Tropenmedizin (BNI), Forschungszentrum Borstel (FZB) und das Heinrich-Pette-Institut (HPI), weltweit anerkannte Einrichtungen für Grundlagenforschung zu globalen Infek tionskrankheiten. Außerdem beteiligt sich das Universitätsklinikum Hamburg-Eppendorf an der Graduiertenschule. Die Förderung stammt aus Mitteln des Paktes für Forschung und Innovation. Die Leibniz-Graduate School bietet den Stipendiaten ein

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20

Die Stipendiaten der Leibniz-Graduate-School mit Dr. Nicole Nolting (li), Prof. Dr. B. Fleischer (BNI, 2.v.re.), Dr. Heinrich Hohenberg (re)

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2121anspruchsvolles, interdiziplinäres Lehr- und Ausbildungspro gramm, in dem sie neben ihrem eigenen Forschungsthema intensive Einblicke in Fragestellun gen und Methoden der Para sitologie, Bakteriologie, Viro logie und Immunologie erhalten. Dabei befassen sich die jungen Wissenschaftler mit hochaktuellen und bedeu-tenden Problemen der Infek tionsforschung.

Neben dem engen Mentoring durch zwei Betreuer und neben regelmäßigen Weiterbildungen in wissenschaftlicher Theorie und Praxis, präsentieren sich die Stipendiaten bei Fortschrittsberichten und Seminaren in englischer Sprache. Des Weiteren wird das Programm ergänzt durch Kurse in Projektentwicklung, Dritt-mitteleinwerbung, Dokumentation, Präsentationstechniken und Wissenschafts-kommunikation. Die Graduierten besuchen wissenschaftliche Tagungen und ver-bringen einen mehrwöchigen Forschungsaufenthalt in einem Labor im Ausland.

Ferdinand-Bergen-Stipendium

Die Ferdinand-Bergen-Stiftung unterstützt seit 2001 die Forschung am Heinrich-Pette-Institut. Seit 2008 fördert die Ferdinand-Bergen-Stiftung darüber hinaus besonders begabte und leistungsbereite Doktoranden der Naturwissenschaften und Medizin im Rahmen eines Ferdinand-Bergen-Stipendiums. Die Stipendiaten erhalten eine dreijährige finanzielle Grundförderung auf Basis einer halben Stelle und werden regelmäßige Workshops des HPI sowie des „Leibniz Center for Infection“ (LCI) besuchen. Nach der Sichtung vieler hervorragender Bewerbungen wurde das Stipendium an Christine Henning aus der Nachwuchsgruppe „Zelluläre Virus abwehr“ vergeben.

Das HPI ermuntert Diplomanden und Doktoranden, verstärkt Fachtagungen zu besuchen, und schafft durch finanzielle Unterstützung einen zusätzlichen Anreiz. Seit Ende 2008 wird ein Reisekosten-Zuschuss von 250 Euro für die aktive Teil-nahme (Poster, Vortrag) an wissenschaftlichen Fachtagungen gewährt, der schon nach kurzer Zeit zu einer erhöhten Teilnahme von vor allem jungen HPI-Mit-arbeitern an internationalen Fachtagungen geführt hat.

HPI-interner Sprachunterricht für deutsche und ausländische Mitarbeiter

Seit 2007 ermöglicht das HPI seinen Mitarbeitern die Teilnahme an ganzjährigen Sprachkursen (für Deutsche: Englisch-Sprachkurse, für Ausländer: Deutsch-Sprachkurse), die im Institut abgehalten werden. Der leistungsgestaffelte Sprach-unterricht soll die englischsprachige Kommunikation innerhalb des HPI ver-bessern. Die Deutschkurse helfen interessierten ausländischen Mitarbeitern, deutsche Spachkenntnisse zu erwerben oder zu erweitern und ihre Kommunika-tion am Arbeitsplatz zu verbessern.

Allgemeiner Überblick

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22Gleichstellungsinitiative am HPIDas 60-jährige Jubiläum des HPI gab Anlass, die Förderung von Frauen als Mit-arbeiterinnen in der Wissenschaft, Verwaltung und Dienstleistung des Instituts weiter zu stärken. Beim „Frauentag am HPI“ am 29. Mai 2008 wurden Ideen und Konzepte hierfür entwickelt. Anregung erhielten die Kolleginnen des HPI durch Sylvie Faverot-Spengler, die Gleichstellungsbeauftragte am DESY, die über ihre langjährige Erfahrung in der Frauenvertretung und Umsetzung von Fördermaß-nahmen am DESY berichtete.

Anlässlich der WGL-Jahrestagung 2008 bekannte sich das Heinrich-Pette-Institut in Form einer Selbstverpflichtung zu den inhaltlichen Zielen der „forschungs-orientierten Gleichstellungsstandards“ der Deutschen Forschungsge mein schaft. Der Vorstand des HPI startete in enger Zusammenarbeit mit der neu gewählten Gleichstellungsbeauftragten, Alicja Iwanski, eine Initiative zur Ver besserung der Familienförderung und Gleichstellung von Frau und Mann am Arbeitsplatz. Ein Ziel dieser Aktivitäten war es, sich am 30. Januar 2009 für das „TOTAL E-QUALITY“ Prädikat zu bewerben. Der Verein „TOTAL E-QUALITY Deutschland“ zeichnet Unternehmen, Organisationen, Hochschulen und For-schungseinrichtungen, die eine an Chancengleichheit orientierte Personalpoli- tik verfolgen, mit diesem Prädikat aus.

Das HPI hat hierfür einen Gleichstellungsplan erarbeitet, in dem unter anderem die Notfallbetreuung für Kinder (0 bis 12 Jahre) gewährleistet wird. Dazu hat das HPI eine Vereinbarung mit der „Vereinigung Hamburger Kindertagesstätten GmbH“ abgeschlossen. Seit Juni 2008 werden alle Beschäftigten, die sich in Elternzeit oder in der Beurlaubung befinden, außerdem auf Wunsch in den allgemeinen Informationsfluss des HPI eingebunden. Das Informationsangebot wird aktiv genutzt.

Die HPI-Gleichstellungsbeauftragte Alicja Iwanski (Mitte) mit ihrer Stellvertreterin Ute Neumann (li) und Leibniz-Präsident Prof. E. Rietschel beim Leibniz-Workshop „Chancengleichheit“

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Das „Leibniz Center for Infection“ (LCI) Das „Leibniz Center for Infection“ ist die erste wissenschaftliche Allianz inner-halb der Leibniz-Gemeinschaft, die Forschungsaktivitäten mehrerer regionaler Leibniz-Institute unter einem Dach vereint. Das Bernhard-Nocht-Institut, das Forschungszentrum Borstel sowie das Heinrich-Pette-Institut nehmen durch die Gründung dieses Forschungsverbundes eine Vorreiterrolle bei der Schaffung neuer wissenschaftlicher und öffentlichkeitswirksamer Konzepte in der Leibniz-Gemeinschaft ein. Sie stärken dadurch die Infektionsforschung im norddeut-schen Raum, reagieren gemeinsam auf aktuelle infektiologische Probleme und Fragestellungen und sichern gleichzeitig ihre internationale Spitzenposition auf diesem Gebiet.

Das LCI koordiniert und unterstützt gemeinsame wissenschaftliche Projekte, ermöglicht eine gemeinsame Öffentlichkeitsarbeit im Bereich der Infektions-forschung und gewährleistet gleichzeitig die Beibehaltung der individuellen Pro- file und Forschungsthemen der beteiligten Institute. Innerhalb eines thematisch zentrierten Leibniz-Zentrums können sich die beteiligten Institute mit mehr Gewicht und fokussierter in regionalen und deutschlandweiten Verbünden präsentieren und zudem in neuen Forschungsallianzen wie Exzellenzclustern, Trans regio-Anträgen, SFBs oder zukünftig auch europaweit in Projekten der EU-Forschungsrahmenprogramme engagieren. Solche Beteiligungen können durch das LCI zentral, längerfristig und auf infektiologische Themen zuge-schnitten organisiert werden und ermöglichen eine institutsübergreifende strategische For schungsplanung, mit dem Potenzial, schnell thematisch passende neue Verbünde zu identifizieren und entsprechend koordiniert aktiv zu werden.

Gemeinsame Projekte des LCI

Seit 2007 finden innerhalb des LCI Workshops statt, die sich mit spezifischen technologischen Schwerpunkten der Institute beschäftigen und so die Schaffung institutsübergreifender Technologieplattformen unterstützen. Hierbei geht es um das Vermitteln und Erlernen von Methoden und Technologien, die für unter-schiedliche infektiologische Fragestellungen genutzt werden können. Dadurch wird nicht nur das wissenschaftstechnologische Arbeits- und Know-how-Spektrum eines jeden Instituts im Rahmen der Nutzung gemeinsamer Ressourcen erweitert. Vielmehr tragen institutsübergreifende Technologieplattformen dazu bei, inter-national kompetitive Hochtechnologien und einen entsprechenden Gerätepark zur Verfügung zu stellen, der z.B. ein weites Spektrum an Imaging- und Screening-verfahren abdeckt.

Aktuell wird das Konzept der gemeinsamen Hochtechnologieplattformen durch die Installation eines tomographischen Cryo-Elektronenmikroskops (CTEM) im HPI umgesetzt. Für die Anschaffung dieses Großgerätes wurden, im Rahmen des „Paktes für Forschung und Innovation“ federführend durch das HPI, in einem durch gemeinsame Projekte der LCI-Partner unterstützten Forschungsantrag Mittel in Höhe von 800T Euro eingeworben. Zukünftige Planungen sehen weitere

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24Kooperationen im Bereich des High-Throughput-Screening, der konfokalen Laser- Scanning Mikroskopie und der Zellsorting-Technologien vor. Auch die Bereit-stellung und Verwendung unterschiedlichster Modellsysteme und Organismen (3D-Zell- und Gewebesysteme, Versuchstiermodelle), verbunden mit entsprechen-der Expertise, sollen zukünftig verstärkt in Angriff genommen werden. Bereits vorhandene Projekte sind in diesem Tätigkeitsbericht ausgewiesen.

Regelmäßige Treffen der Entscheidungsträger der einzelnen Institute stellen sicher, dass neben den institutsspezifischen wissenschaftlichen Strategien eine über-greifende LCI-Strategie existiert, die sich vor allem mit den Fragen gemeinsamer Förde rungen und verstärkten Zusammenarbeiten beschäftigt. Die gegenseitige Beteiligung der Partner an Beiratsbegehungen unterstützt dieses Vorhaben. Aus dieser Strategie heraus haben sich bereits kürzlich gemeinsame SFB-Anträge sowie Anträge um Mittel der Exzellenzclusterinitiative der Freien und Hansestadt Hamburg ergeben. Mittel- und langfristig sollen durch die Abstimmung gemein-samer LCI-Konzepte einerseits Wettbewerbsvorteile für die einzelnen Institute im Bereich der Drittmitteleinwerbung entstehen, andererseits sollen die Zuwendungs-geber einen in der Infektionsforschung breit aufgestellten, aber themenfokussier-ten Verbundpartner in der Infektionsforschung vorfinden. Dieser Verbundpartner ist mit zukunftsorientierten Konzepten, exzellenten Projekten, modernsten Tech-nologieplattformen und Modellsystemen und auch im internationalen Maßstab ausgewiesener Exzellenz eine gute Adresse für Fördermaßnahmen im Bereich der Erforschung weltweit relevanter humanpathogener Infektionskrankheiten.

Zukunftsperspektiven

Projekte, die die Zusammenarbeit von Wissenschaftlern der LCI-Institute in Zukunft noch verstärken können, sind beispielsweise LCI-Veranstaltungen wie Retreats, Kooperationsworkshops und gemeinsame thematische Tagungen. Es ist mit Sicherheit zu erwarten, dass die Leibniz-Graduate School sowie die zuneh-mende Zusammenarbeit einzelner Forschungsgruppen und beispielsweise ein institutsübergreifender Publikationspreis für junge Wissenschaftler zu weiteren gemeinsamen Forschungs- und Planungsinitiativen der beteiligten LCI-Partner führen werden.

In diesem Sinne ist für die Zukunft vor allem ein gemeinsames öffentliches Auf-treten der Leibniz-Partnerinstitute geplant. Zu diesem Zweck soll im Sommer 2009 in einem ersten Schritt eine detaillierte Informationsbroschüre erscheinen, die nicht nur über das LCI insgesamt, sondern auch über bisherige Zusammen-arbeiten und gemeinschaftliche Projekte berichtet. Ergänzt wird die Broschüre voraussichtlich im Herbst durch einen Internetauftritt des LCI, der vor allem auch noch einmal die „Graduate School“ darstellt und hier den Studenten als Informations plattform für die Ankündigung und Planung von Veranstaltungen dient. Daneben sollen hier aktuellste wissenschaftliche Höhepunkte der Institute sowie die Dar stellung der gemeinsamen Technologieplattformen und der Ko- operationen zu finden sein.

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Eine der am weitesten in die Zukunft reichenden LCI-Aktivitäten in der norddeut-schen Wissenschaftslandschaft betrifft die Beteiligung des LCI am Zentrum für strukturelle Systembiologie (Centre for Structural Systems Biology, CSSB), das ab 2010/2011 auf dem DESY-Campus geplant ist. Ziel dieser Großforschungs-einrichtung mit infektionsbiologischem Schwerpunkt ist das Erreichen eines möglichst umfassenden und detaillierten Verständnisses der diversen molekularen Wechselwirkungen innerhalb lebender Zellen und Gewebe im Rahmen struktur- und systembiologischer Grundlagenforschung. Wichtige Grundlage hierfür ist die systematische Analyse und Modellierung der molekularen Architektur und Dynamik biologischer Systeme über sämtliche Auflösungsskalen. Es ist geplant, dass das LCI sich mit einer gemeinsamen Arbeitsgruppe an dieser interdisziplinären Einrichtung beteiligt. Daneben werden weitere Forschungsabteilungen von Universitäten und außeruniversitären Institutionen gemeinsam mit dem DESY dort angesiedelt wer-den, die komplexe zelluläre Prozesse im Infektionsgeschehen im Rahmen eines systembiologischen Gesamtkonzepts untersuchen sollen. Auf Grund der immensen medizinischen Bedeutung der Infektionsforschung ist geplant, frühzeitig Partner aus der pharmazeutischen Industrie mit einzubinden.

Mit der Errichtung des CSSB als (über)regionalem Forschungscampus wird im Großraum Hamburg ein weithin sichtbarer „Leuchtturm“ in der biomedizinischen Forschung mit einem Schwerpunkt in der Infektionsforschung entstehen – eine optimale Gelegenheit für das LCI, sich nachhaltig zu profilieren und über eine Zusammenarbeit mit weiteren Infektionsforschern, Systembiologen, Struktur-biologen und Physikern eine internationale Spitzenposition in der Infektionsfor-schung einzunehmen. Das frühzeitige Engagement des LCI im CSSB wird von Seiten der Leibniz-Gemeinschaft befürwortet und die zukünftige Gründung einer gemeinsamen Forschungsabteilung von Seiten des Präsidiums aktiv unterstützt.

Das „Leibniz Center for Infection“, offiziell gegründet im Jahr 2005, durchläuft eine zunehmend dynamische Entwicklung. Die bisher gemeinsam durchge-führten Projekte und eingeworbenen Mittel zeigen, auf der Basis einer verstärkt koordinierten Arbeit, bereits Erfolge. Durch die Schaffung einer halben Stelle für die Betreuung der Interessen der beteiligten Partnerinstitute und die gemein-same Öffentlichkeitsarbeit stehen seit Mitte 2008 personelle Ressourcen zur Verfügung, die den gesteigerten Anspruch des LCI an nationaler Sichtbarkeit und wissenschaftlicher Exzellenz auf der organisatorischen Seite unterstützen kön-nen. Trotz der individuellen Forschungsausrichtung jedes Instituts und seines spezifischen „Institutscharakters“, der sich über viele Jahrzehnte hinweg ent-wickelt hat, verbindet das BNI, das FZB und das HPI nicht nur die weithin über die Grenzen Deutschlands hinweg anerkannte wissenschaftliche Leistung zum Thema „Infektionsforschung“, sondern auch der „Leibniz-Gedanke“, medizi-nische Grundlagenforschung im Bereich der weltweit wichtigsten und gefähr-lichsten humanpathogenen Infektionskrankheiten im Hinblick auf gesamtstaat-liche Gesundheitsverantwortung zu betreiben. Wir sind zuversichtlich, dass sich das LCI in Anbetracht dieser Gemeinsamkeiten langfristig zu einer über regional bekannten Leibniz-Einrichtung und zu einem internationalen Forschungs-schwer punkt für infektionsbiologische Fragen entwickeln wird.

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IntroductionAccording to the World Health Organization (WHO), globally infectious diseases are by far the leading cause of death. The ongoing HIV/AIDS-pandemic gives an example for the disastrous socio-economic impact that a single infectious agent may exert on developing countries, as well as for the financial burden that is placed on the public health-systems in the industrialized world. The detailed investigation of the life-cycle of infectious agents is therefore of utmost impor-tance for the successful development of novel and improved anti-microbial strategies.

Within this research program the life-cycle of various humanpathogenic viruses, or related animal viruses, are investigated in detail. For example, these include Hepatitis B Virus (HBV), human and simian immunodeficiency virus (HIV and SIV), Herpes Simplex-Virus (HSV), Epstein-Barr Virus (EBV), Kaposi-Sarcoma associated Herpes Virus (KSHV), Simian Virus 40 (SV40), Merkel Cell Polyomavirus (MCPyV), Adenoviruses and various murine retroviruses.

Viruses are obligatory parasites that can produce progeny only after successful infection of permissive host cells. Thus, virus replication depends on multiple and frequently critical interactions involving viral and cellular components. Because viruses exploit preexisting cellular pathways, they also serve as valuable tools to understand the molecular basis of fundamental host cell mechanisms such as, for example, the regulation of immune responses or the induction of cell stress and carcinogenesis. Therefore, the investigation of different viruses and their interac-tion with their host cells will not only improve our understanding of the different strategies that are applied by the respective viruses to secure progeny formation, but will also explicitly expand our knowledge on cellular metabolism. Finally, these studies will also reveal novel viral and cellular target structures, that may be subsequently exploited for the development of advanced antiviral therapies.

As in previous years, the major research activities in 2007-2008 within the pro-gram area “Virus-Host Interaction” can be roughly divided into research fields that focus on early and late aspects of virus infection. Examples for the first research area are the analysis of the receptor usage of gammaretrovirus envelope proteins or of the host tropism and entry mechanisms of Hepatitis B Viruses. Examples for the latter research area are studies of the mechanisms that direct the nuclear export of adenoviral and lentiviral mRNAs, on Adenovirus- and SV40-induced transformation and tumorigenesis, on the activity of Polyomavirus and Herpes Virus encoded microRNAs, or on the assembly and budding of Hepatitis Viruses. Furthermore, these projects were complemented by analyses that, for example, investigated SV40-triggered immune responses or HIV-1-induced pathogenesis, as well as by the development of novel experimental antiviral therapies. A detailed description of the individual projects is given further below.

Within the report period significant progress with respect to improved network-ing, i.e. by moving away from individual towards inter-institutional research

Programmbereich „Virus-Wirts-Wechselwirkungen“Program Area „Virus-Host Interactions“Head: Prof. Dr. Joachim Hauber

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Virus-Host Interaction

projects, was made. Particularly the interdisciplinary consortium “Combating Drug Resistance in Chronic Myeolid Leukemia and HIV-Infection”, which is funded by the Federal Ministry of Education and Research (BMBF), was initiated by a project originating in the program area “Virus-Host Interaction”. This con-sortium represents an outright translational research approach and integrates laboratories from the University Hospital Hamburg-Eppendorf, the Universities of Hamburg and Lübeck and the Heinrich-Pette-Institute. By bringing together preclinical and clinical virus research, biochemistry, structural biology, chemistry and bioinformatics into a close collaboration, this consortium may also serve as a model for future proposals within the framework of the upcoming initiative for excellence in the sciences of the state of Hamburg (Landesexzellenz-Initiative).

In December 2007 the new junior research group “Virus Pathogenesis”, headed by Dr. Michael Schindler, became operational and the program area “Virus-Host Interaction” significantly benefitted from this fact. Dr. Schindler’s research pri-marily focusses on how HIV-1 induces pathogenesis in the infected organism and how viral factors interfere with host cell components to counteract antiviral host activities. By recruiting this internationally recognized scientist the Heinrich-Pette-Institute continued to refocus its direction of research according to its mis-sion statement towards the investigation of the biology of human viruses, the pathogenesis of virus-induced diseases and antiviral mechanisms of the infected organism.

While the main load of the straight-forward virus-related projects are clearly investigated within the program area “Virus-Host Interaction”, it has to be noted that many projects are closely connected, experimentally as well as intellectually, with projects of the program area “Cellular Dysregulation”. Finally and as docu-mented in the publication list, an increased research output for the 2007-2008 period within the program area “Virus-Host Interaction” is ultimately reflected in numerous publications in peer-reviewed internationally recognized scientific journals, as well as by various patent applications that originated from our inves-tigation of critical virus-host interactions. The latter particularly highlights a continuous increase in translational research conducted within this program area, in turn resulting in significantly increased third-party research funds.

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Research ProjectsHelper function of wild-type p53 in Simian virus 40 induced cellular transformation

A. Hermannstädter, C. Ziegler, M. Kühl, G.V. Tolstonog, W. DeppertHeinrich-Pette-Institute, Hamburg, Germany

Abortive infection of matched pairs of mouse fibroblasts differing in p53 gene status (p53+/+ cells vs. p53-/- cells) with SV40 revealed a strongly decreased efficiency for colony formation in soft agar in p53-/- cells com-pared to p53+/+ cells. Transformation competence of SV40 in p53-/- cells could be fully restored by reconstituting wild-type p53 (wtp53), but not mutant p53 (mutp53) expression. The helper effect of wtp53 is mediated by its cooperation with large T antigen (LT), as stable expression of LT in p53+/+ cells resulted in full transformation, while LT expression in p53-/- cells only enhanced saturation density, but could not promote growth in suspension or

Gene expression analysis in 3T3 LT, 3T3 IG, 10 1 LT and 10 1 IG cells. A: Bar diagram summarizes the total number of genes up- or down-regulated in 3T3 LT and 10-1 LT cells in comparison to 3T3 IG and 10-1 IG cells, respectively. Venn diagram illustrates the overlap between the lists of regulated genes. B: Fold change in Igf1 gene expression relative to non-LT expressing cells. Total RNA isolated from two biological replicates of 3T3 LT, 3T3 IG, 10 1 LT and 10 1 IG cells was analyzed by Real-time PCR with primers specific for Igf1 and Gapdh. Each assay was done in triplicate and expression of Igf1 was normalized to Gapdh. Igf1 expression in LT-expressing cells is reported relative to control cells (3T3 IG, 10 1 IG). Control cells were set at 100% and calculations revealed following results for Igf1 expression in p53+/+ cells (3T3 LT) 16.1% (+/- 2.3/2.6%) and in p53-/- cells (10 1 LT) 80.2% (+/- 1.5/1.5%). Calculation of error bars (RQ Min/RQ Max) is based on confidential level of 95%. C: Table presents the list of ECM (extracellular matrix proteins)-encoding genes down-regulated in 3T3 LT cells (> 2-fold) comparing to the control 3T3 IG cells. Gene expression values were obtained using Agilent Whole Mouse Genome Microarray chips.

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in soft agar to a significant extent. The p53 helper function resides in the p53 N-terminus, as an N-terminally truncated p53 (ΔNp53) could not rescue the p53-null phenotype. The p53 N-terminus serves as a scaffold for recruiting transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex. Consequently, LT affected global gene expression in p53+/+ cells significantly stronger than in p53-/- cells. In particular, genes related to the expression of a transformed phenotype, like genes encoding extracellular matrix proteins were specifically down-regulated in 3T3 LT cells (p53+/+), but not in 10-1 LT cells (p53-/-). Furthermore, we were able to demonstrate that the Igf1 gene, a presumed target gene of the LT-p53 complex, is specifically down-regulated only in 3T3 LT cells, but not in 10-1 LT cells. Our data suggest that recruitment of transcriptional regulators into the LT-p53 complex serves to modify cellular gene expression to the needs of cellular transformation.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6

Role of c-Met in SV40-induced mammary tumorigenesis

S. Babu (1), C. Heinlein (1), F. Krepulat (1), B. Otto (2), T. Streichert (2), G.V. Tolstonog (1), W. Deppert (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry, Hamburg, Germany

We generated and characterized a transgenic mouse model (WAP-T mice) suit-able for molecular analysis of initiation and progression of mammary adenocar-cinoma. Expression of SV40 early proteins in WAP-T mice is initiated during late pregnancy, and tumors develop from T-antigen expressing mammary epithelial progenitor cells that survived involution. Molecular analyses of the tumors by aCGH and gene expression profiling revealed that the Met gene is amplified and strongly expressed in nearly all undifferentiated tumors. Therefore, we focused on the role of c-Met during SV40-induced tumorigenesis.

Double staining for c-Met and SV40 Large T-antigen (LT) in a mammary epithelial tumor cell line established from a WAP-T tumor showed that c-Met expression levels were directly proportional to LT expression levels, indicating a role for LT in regulating Met expression. Ectopic expression of LT in mouse embryo fibroblasts with wild-type p53 (wtp53) or p53-null background revealed a significant LT-dependent increase in c-Met expression in cells with wtp53 compared to p53-null background. The data suggested that c-Met expression is repressed by wtp53, and that this repression is alleviated by p53 inactivation through complex formation with LT. To confirm the role of wtp53 in c-Met expression, we investigated c-Met expression in mouse cG9 cells which express a temperature-sensitive p53 that is wild-type at 32°C and mutant at 39°C, respectively. We observed increased expression of c-Met when p53 was in a mutant conformation. The data further supported the postulated p53-dependent repression of the Met gene and the action of LT that leads to transcriptional inactivation of p53 by complex formation. By ChIP assay we detected binding of p53 to the Met promoter, which was abolished

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Met gene amplification and expression in SV40-induced mouse mammary carcinoma. A: UCSC Genome Browser view of mouse Met genomic loci on the chromosome 6. B-C: Graphic visualization of gene copy number and expression data. Cellular RNA and genomic DNA from tumor samples were analyzed using Affymetrix gene expression microarrays (C) and Agilent whole mouse genome microarrays (B), respectively. A total of 65 samp-les including SV40-induced mammary carcinomas in mono- (SV40 early region: WAP-T, WAP-NP8) and bi-trans-genic mice (SV40/mutp53: WAP-T-H22, WAP-NP8-W10) as well as spontaneous breast tumors were submitted to the molecular analysis. Expression values were standardized by the z-score method. Array-CGH values are log2 ratios after segmentation. Most of the poorly differentiated tumors (grades G3 and G4) are characterized by amplification of Met loci. The amplico boundaries greatly vary between individual tumors. The asymmetrical distribution of scale of values highlights the strength of gene amplifications. The expression values correlate with the amplifications.

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when LT was co-expressed. Silencing of c-Met using shRNA in LT transformed cells abrogated the cells’ ability to form colonies in soft agar, highlighting the role of c-Met in proliferation and substrate-independent growth. We conclude that p53 acts as a repressor of the Met gene. Alleviation of this repressor function by LT is essential for LT-mediated cellular transformation.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6

Mutant p53 gain of function in mammary carcinogenesis

E. Lenfert (1), C. Heinlein (1), F. Krepulat (1), T. Streichert (2), B. Otto (2), W. Deppert (1), G.V. Tolstonog (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry, Hamburg, Germany

Missense mutations and loss of the wild-type allele in the TP53 gene frequently accompany tumor formation. The most prevalent missense mutations found in human breast cancer are at the positions R248 and R273. The p53 mutant allele encodes a protein (mutp53), which has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but has retained the propensity to interact specifically with nucleic acids and proteins.

To study the influence of mutp53 on the development of SV40-induced mouse mammary carcinoma we developed bi-transgenic WAP-T (SV40 early region) x WAP-mutp53 mouse models with equivalent mutations (R245W, R270H). Mutp53 acts synergistically with the SV40 T/t-antigens, enhances tumor incidence and aggravates development of undifferentiated carcinomas and pulmonary metastasis.

To dissect the molecular mechanisms of mutp53 function we performed gene expression profiling of tumor and normal tissues. By bioinformatic evaluation of microarray data we identified a small set of genes which expression strongly correlates with mutp53. We focused on Apobec3 gene and found that the tumors and involuted glands of WAP-T-mutp53 bi-transgenic animals con-tained more Apobec3-positive cells than the respective tissues of the WAP-T mono-transgenic mice. These preliminary results indicate a role of Apobec3 in tumor progression and aggressiveness. To test the hypothesis that mutp53 may influence miRNA expression and in this way provide an additional level of regulation, miRNA expression in tumor samples of 4 mono-transgenic and 4 bi-transgenic mice was quantified on Agilent chips. Analysis of miRNA expression profiles pointed at functional crosstalk between wild-type and mutp53, and supported the possibility that mutp53 enhances metastasis via changes in miRNA expression.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6

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A preclinical model for monitoring treatment and prevention of metastasis in mammary carcinoma

C. Mänz (1), F. Wegwitz (1), A. Diesterbeck (1), A. Preuß (1), J.S.D. Mieog (2), G.-J. Lieffers (2), W.G.M. Löwik (2), F. Alves (3), G. Tolstonog (1), W. Deppert (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Leiden University, Leiden, The Netherlands (3) University of Göttingen, Germany

Breast cancer is the most frequently diagnosed type of cancer and the second leading cause of death among women in the Western world. Once localized breast tumors spread to other organs (metastasis), breast cancer is often incurable. The WAP-T and WAP-T x WAP-mutp53 mice developed in our lab adequately model the slow onset and development of human breast cancer from ductal carcinoma in situ (DCIS) up to invasive growth with metastasis. With these mice we aim to mirror the clinically relevant situation of metastatic disease that arises after excision of the primary tumor.

We have established a cell line from an endogenous, undifferentiated mammary carcinoma of a bi-transgenic WAP-T-WAP-mutp53 mouse which exhibits char-acteristics of tumor stem cells (GeTo cells). Orthotopic transplantation into syngeneic immune-competent mice of < 100 GeTo cells leads to the develop-ment of tumors with similar characteristics as primary tumors in WAP-T mice. Tumors arising from transplanted GeTo cells into WAP-T mice dissemination into a variety of tissues, blood and bone marrow. Disseminated tumor cells can be detected with a sensitivity of less than 1 in 105 cells. Some transplanted ani-mals develop visible lung metastases already within the period of tumor growth. To mimic the situation encountered by human mammary carcinoma patients, primary tumors arising from transplanted GeTo cells are surgically removed at an early stage to allow more time for disseminated cells to establish second site malignancies, which in human patients can take up to ten years. This preclinical model can be used to test various treatment regimens for the elimination of disseminated tumor cells and thus for prevention of metastasis in collaboration with clinical partners. To further refine the analysis, state of the art in vivo imaging of mice will be performed in collaboration with Prof. W.G.M. Löwik, Leiden University, Leiden, The Netherlands and Dr. F. Alves, University of Göttingen, Germany.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and by EU FP6

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Induction of a cellular immune response against SV40 induced mammary carcinoma in transgenic mice

M. Bruns (1), G. Pilnitz-Stolze (1), A. Diesterbeck (1), A. Düsedau (1), O. Utermöhlen (2), U. Schumacher (3), W. Deppert (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institute of Immunology, University of Cologne, Germany (3) Institute of Anatomy II, UKE, Hamburg, Germany

The immune system can also recognize cancer cells as foreign. In order to study the different facets of immune surveillance during tumor progression, transgenic BALB/c mice were established containing the whey acidic protein (WAP) pro-moter upstream of the large tumor antigen (T-Ag) of simian virus 40 (SV40) (WAP-T lines). Upon induction of the transgene, these mice develop endogenous mammary carcinomas, which derive from T-Ag expressing mammary epithelial progenitor cells that survive involution. As BALB/c mice are considered to be “low responders” towards SV40 T-Ag, a 33bp oligomer of the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV), encoding the MHC class I H-2d restricted T-cell epitope, was inserted into the C-terminal coding region of T-Ag (WAP-T-NP lines). This NP-epitope mediates the strong immune reaction against LCMV in BALB/c mice. Expression of the hybrid gene thus allows the analysis of the immune response after LCMV infection of WAP-T-NP mice. While the WAP-T-NP lines are tolerant against SV40, they are not tolerant against LCMV, as these mice mount a strong cellular immune response against the LCMV-NP epitope upon infection. Likewise, adoptive transfer of immune competent cells from LCMV-infected BALB/c wild-type mice into WAP-T-NP mice strongly reduced the number of T-Ag positive cells not only in lactating mammary glands but also in cancerous tis-sues. The data point towards an immune surveillance, where NP epitope-specific CD8+ T-cells not only are capable of virus elimina-tion, but can also efficiently reduce the number of T-Ag expressing tumor cells in WAP-T-NP mice. The data provoked the question, why this strong immune reaction is not mounted during endog-enous tumor development. As a possible explanation we analyzed whether this phenomenon might relate to the observation that chronically LCMV-infected mice contain only functional impaired virus-specific CD8+ T-cells (“exhausted” T-cells), which have

Comparison of PD-L1-treated (left) and untreated WAP-T-NP8 mice (right). Comparison of WAP-T-NP8 mice treated either with antibod-ies against PD-L1 (right) or left untreated (left). Formalin-fixed and paraffin embedded sections of mamma carcinomas were stained by antibodies against SV40 T-Ag (above) or PD-L1 (below).

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selectively up-regulated the programmed death 1 (PD-1) protein. As demonstrated for LCMV-carrier mice, in vivo application of antibodies that blocks the interac-tion of this receptor-molecule on T-cells with its ligand PD-L1 indeed led to an elimination of T-Ag expressing tumor cells in WAP-T-NP mice. Consequently, it was found by FACS and immune histological studies that antibody-treatment of tumor mice with the aim to inhibit the binding of PD-1 to its ligand revealed a strong reduction of PD-1+ immune cells and at the same time resulted in a successful elimination of T-Ag and PD-L1 expressing epithelial cells in parallel (Fig. 1). This finding could provide a powerful new strategy for the elimination tumor cells via re-vitalization of exhausted immune cells by blocking the PD-1/PD-L1 interaction.

*Supported by EU FP6

Steroid dependent control of HIV replication

K. Wiegers, D. Schwarck, R. Reimer, W. BohnHeinrich-Pette-Institute, Hamburg, Germany

Nuclear transport and integration of the HIV genome require the functional interaction between cellular and virus associated proteins. This interaction has significant influence on the efficiency of virus replication. In resting peripheral mononuclear blood cells (PBMCs) infection with HIV-1 is not productive due to a block that occurs early after infection prior to provirus formation. We noticed that in resting PBMCs the status of the glucocorticoid receptor (GR) contributes to the restriction in virus replication. Treating PBMCs with a GR specific ligand is sufficient to overcome the block and increases the proportion of integrated provirus. The ligand mediated effect is only seen when the ligand is given early after infection. In addition, it requires the presence of the viral protein Vpr. Vpr is associated with the pre-integration complex and known to bind the nuclear receptor. The data suggest that the block is based on an inefficient nuclear trans-port and/or impaired integration of the virus genome. Microscopic data show that in unstimulated, naïve PBMCs endogenous GR is retained in the cytoplasm, as is also an ectopic Vpr expressed in these cells. Stimulation with a GR specific ligand translocates both proteins into the nucleus. Thus, cytoplasmic retention of the HIV preintegration complex by the non-activated nuclear hormone receptor seems to be the obstacle for efficient HIV replication in naïve PBMCs. Glucocorticoid treatment overcomes this restriction by stimulating nuclear transport of the receptor. In accordance, in a cell line with a constitutive cytoplas-mic GR efficient proviral integration requires addition of a GR ligand. The data suggest that steroids overcome the restriction on HIV provirus formation and thereby increase the reservoir of virus producing cells.

*Supported in part by Deutsche Forschungsgemeinschaft (DFG)

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Control of viral mRNA export by adenovirus regulatory proteins

O. Koyuncu (1), K. Kindsmüller (1), P. Blanchette (2), P. Groitl (1), F. Dallaire (2), T. Speiseder (1), P. Branton (2), J. Hauber (1), T. Dobner (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) McGill University, Department of Biochemistry, Montreal, Canada

Human adenoviruses have small linear double-stranded DNA genomes encoding only the virus structural polypeptides and a limited number of regulatory pro-teins. In order to be able to maintain a compact genome size, and hence restrict-ed coding capacity, yet remain an effective pathogen these viruses have evolved efficient strategies to utilize the host cell machinery for viral gene expression, DNA replication and formation of progeny virions. Their ability to subvert host macromolecular synthesis at defined stages of the viral replication cycle is the result of both the viral program of transcriptional regulation and virus-encoded functions that control mRNA processing, nucleo-cytoplasmic mRNA transport and translation. It is the posttranscriptional mechanisms in particular which serve to facilitate production of large quantities of viral structural polypeptides and to shut-off host cell protein synthesis at late times of infection. In produc-tively infected cells regulated viral mRNA splicing and viral mRNA export is controlled by at least three adenovirus proteins E4orf6, E1B-55K and L4-100K.

Ad5 E1B, E4 and L4 proteins are multifunctional regulators of virus replication. Ad E1B, E4 and L4 gene prod-ucts encompass a diverse collection of functions that modulate viral and cellular growth control at all levels of the gene expression pathway. In productively infected cells E1B directly or indirectly alters the pattern of splicing and stability of late viral RNAs. In addition E1B and E4 proteins been implicated in the preferential nuclear export of viral late RNA transcripts, inhibition of cellular mRNA transport and protein synthesis, while L4-100K shuts off host cell translation, promotes translation of tripartite leader (TL) containing viral late mRNAs via ribosome. In addition the E1B/E4orf6 complex enhances the degradation of cellular target proteins on cytoplasmic and/or nuclear proteasomes.

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These directly or indirectly alter the pattern of splicing and stability of late viral RNAs and have been implicated in the preferential nuclear export of viral late RNA transcripts, inhibition of cellular mRNA transport and protein synthesis, selective translation of tripartite leader (TL) containing viral late mRNAs via ribosome shunting and nuclear localization of Ad5 capsid components, such as hexon. In addition the E1B-55K/E4orf6 complex promotes the proteasomal degradation of the tumor suppressor protein p53, DNA Ligase IV and Mre11, a subunit of the Mre11/Rad50/NBS1 (MRN) DNA double-strand break repair complex. The latter activity may also involve nuclear depletion and cytoplasmic deposition of p53 and MRN components into aggresomes, which, consistent with their role in the removal of aggregated proteins from the cytosol, further accelerates proteolytic degradation of MRN subunits. Collectively available data suggest that most if not all of the lytic functions ascribed to E1B-55K, E4orf6 and L4-100K during infection including control of viral mRNA export and translation are achieved through continuous nucleo-cytoplasmic shuttling and interactions with a variety of host cell factors mediating nuclear import and export processes.

Efforts to identify cellular targets that link the E1B-55K, E4orf6 and L4-100K to nuclear export and import pathways showed that each of the viral proteins pos-sesses a single leucine-rich nuclear export signal (NES) of the HIV-1 Rev-type and can individually shuttle between the nuclear and cytoplasmic compartment via the export receptor CRM1. In addition, nucleocytoplasmic trafficking of E1B-55K involves a CRM1-independent nuclear export pathway, which is controlled by covalent conjugation of the small ubiquitin-related modifier protein 1 (SUMO1). Thus, SUMO1 conjugation and deconjugation provides a molecular switch that commits E1B-55K to CRM1-dependent and -independent export pathways. E1B-55K, E4orf6 and L4-100K also interact with cellular factors that could bridge the viral protein complex to other cellular export receptors, such as HuR, pp32, APRIL and/or TAP/NXF1. Nonetheless, the contribution of these cellular factors to nucleo-cytoplasmic shuttling, selective viral late RNA export and viral late gene expression is still unknown.

As part of our studies of E1B-55K/E4orf6 function during produc-tive infection we found that E4orf6 connects E1B-55K to components of a cellular E3 ubiquitin ligase, thereby allowing the proteasomal degradation of p53, Mre11 and DNA Ligase IV. E1B-55K associ-ates with this high molecular weight complex where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. These observa-tions and the finding that nearly all of E1B-55K present in infected

Nucleo-cytoplasmic shuttling of E1B-55K is regulated by CRM1 and SUMO1-conjugation. Subcellular distribution of wt (GFP-E1B and pE1B-55K) and mutant (pE1B-NES) E1B proteins in the absence or presence of the CRM1 inhibitor LMB. Magnification x 7600.

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cells is found associated with E4orf6 raises the possibility that nucleo-cytoplasmic trafficking and control of mRNA export requires degradation of cellular target pro-teins by the E1B-55K/E4orf6 E3 ubiquitin ligase activity. We have tested this hypoth-esis through the generation and phenotypic analysis of a virus mutant in which the E4orf6 product is unable to form the ligase complex and indeed have found that this mutant behaves identically to an E4orf6minus virus in production of late viral pro-teins, growth, and in the export of late viral mRNAs. Thus, preferential nuclear export of viral late RNA transcripts and simultaneous inhibition of cellular mRNA transport is intimately linked to the E1B-55K/E4orf6 E3 ubiquitin ligase activity.

CRM1-mediated nuclear export of Ad5 L4-100K is required for efficient virus growth. (A) Top: Multiple amino acid sequence alignment of L4-100K polypeptides from human Ad serotypes, subgroups A-D. Numbers in brackets refer to amino acid residues. Residues that are identical in all serotypes are shown in light grey boxes and the conserved hydrophobic residue is marked with a dark grey box (Φ: hydrophobic amino acid residue, X: any amino acid residue). Bottom: The amino acid sequence of wt virus H5pg4100 and the NES virus mutant H5pm4165 with alanine substitutions at positions 383, 386, 390, 392. (B) Virus yield. A549 cells were infected with wt H5pg4100 or H5pm4165 at indicated multiplicities. Viral particles were harvested at indicated time points (h p.i.) and virus yield was determined by quantitative E2A-72K immunofluorescence staining on K16 cells. The results represent the averages from three independent experiments. Error bars indicate the standard error of the mean. (C) A549 cells were infected with H5pg4100 (panel a) and H5pm4165 (panel b) viruses at a multiplicity of 10 FFU per cell. Infected cells were fixed at 36 h post infection (h p.i.) and stained with rat mab 6B10. In all panels nuclei are indicated by a dotted line. Magnification x 7600.

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A highly interesting result was also obtained when we analyzed the contribution of CRM1-mediated nuclear export of L4-100K to productive infection. Phenotypic analyses of a virus mutant (H5pm4165) carrying amino acid exchanges in the highly conserved NES of L4-100K in different human tumor cell lines revealed that it is severely defective in late viral gene expression and virus production compared to the wild-type virus H5pg4100. Localization of the 100K NES mutant was restricted to the nuclei of the infected cells. A similar result was obtained when CRM1 was inactivated by leptomycin B (LMB). These data show that nuclear export of 100K is required for maximal virus growth and suggest that CRM1 is the major export receptor for L4-100K in virus-infected cells. Remarkably, these studies also revealed that L4-100K is a substrate of the protein arginine methylation machinery an emergent post-translational modification system involved in a growing list of cellular processes, including transcriptional regula-tion, cell signaling, RNA processing and DNA repair. L4-100K arginine methyla-tion involves protein arginine methyltransferase 1 (PRMT1), which asymmetri-cally dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. Arginine to glycine exchanges in the RGG boxes significantly diminish 100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation is an important host-cell function required for efficient adenovirus replication.

Taken together, results from studies demonstrate for the first time that efficient late adenoviral gene expression is dependent on PRMT1 and the export receptor CRM1, the latter being most critical for efficient virus growth in human tumor cell lines. In fact, these data suggest substantial mechanistic similarities between adenovirus and complex retroviruses in the control of viral RNA export.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Identification of a new substrate of the adenovirus/E1B-55K/E4orf6 ubiquitin ligase complex

F. Dallaire (1), P. Blanchette (1), P. Groitl (2), T. Dobner (2), P. Branton (1)(1) McGill University, Department of Biochemistry, Montreal, Canada (2) Heinrich-Pette-Institute, Hamburg, Germany

The human adenovirus E4orf6 and E1B-55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains Elongins B and C and Cullin family member Cul5. In addition to p53, its first known substrate, the E1B-55K/E4orf6 complex was shown to promote the degradation of Mre11 and D0NA Ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seem likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and simultaneous inhibition of host cell mRNAs export from the nucleus. In an attempt to identify new E1B-55K/E4orf6 substrates we under-took a proteomic screen using human p53-null, non-small cell lung carcinoma

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H1299 cells expressing either E4orf6 protein alone or in combination with E1B-55K through infection. One cellular protein that appeared to be degraded by E1B-55K in combination with the E4orf6 protein was a species of molecular mass ∼130kDa that was identified as integrin alpha 3 subunit (Very Late Antigen 3 alpha subunit or VLA-3α). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions in the very late phase of the infectious cycle.

*Supported by Canadian Institutes of Health Research – CIHR

Human adenovirus E4 region orf3/4 protein is required for efficient virus replication

T. Speiseder (1), P. Groitl (1), T. Dobner (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

Human adenovirus early region 4 (E4) encodes at least six distinct polypeptides, defined as E4orf1 to E4orf6/7 according to the order and arrangement of the corresponding open reading frames (orfs). E4 gene products operate through a complex network of protein interactions with key viral and cellular regulatory components involved in transcrip-tion (E4orf6/7), protein degrada-tion, nucleo-cytoplasmic trans-port and DNA repair (E4orf6), as well as host cell factors that regu-late cell signaling (E4orf1), post-translational modifications (E4orf4) and the integrity of PML-containing nuclear bodies (E4orf3).

In the context of our studies of E4 function we generated a set of mutant viruses carrying point mutations in the E4 genes. The phenotypic characterization of these mutants revealed that lesions affecting individual orfs had no or only moderate effects on lytic virus growth. By contrast, Ad mutants that lack the entire E4 region or fail to express the E4orf6 protein were severely restricted for growth. Unexpectedly, a virus mutant unable to express E4orf3 plus E4orf4 was as defective as the E4-minus mutant viruses. Further,

Ad2/5 E4 transcription unit. Top: ORF maps spanning the entire E4 region of Ad5. The three reading frames (1, 2 and 3) of the E4 region sense strand from position 35606 (cap-site) to 32802 (polyA-signal) are shown. Gray shaded regions indicate ORF1 to ORF7 coding sequences. Vertical lines bisecting each reading-frame indicate stop codons, whereas small triangles denote methionine codons. Middle: E4 mRNAs are indicated by lines and arrows; introns are indicated by spaces and coding regions are indicated by open rectangles. With the exception of the ORF3/4 mRNA other mRNAs species which have not been detected [Dix, 1993 #76] are not included. Late and Early at the right side denote the temporal class of each individual mRNA species. Bottom: the location of the ORFs and the splice donor/acceptor combinations relative to their position in the Ad5 nucleotide sequence. Abbreviations: ITR, inver-ted terminal repeat; cap, cap-site; poly(A), polyadenylation signal.

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more detailed genetic and biochemical analyses demonstrated that the E4orf3/E4orf4 double mutant exhibits a pleiotropic phenotype and fails to produce the E4orf3/4 fusion protein, which consists of the N-terminal 33 amino acid residues from orf3 and the C-terminal 28 amino acid residues from orf4. Consistent with this a virus mutant lacking E4orf3/4 and E4orf4 replicated to levels nearly equal-ly reduced to the E4-minus mutant. In sum, these results introduce a function for E4orf3/4 and suggest that E4orf3/4 and E4orf4 provide redundant functions for efficient viral replication, which is intimately linked to their identical C-terminal regions.

*Supported by Wilhelm Sander-Stiftung

Localization and importance of the adenovirus E4orf4 protein during lytic infection

M-J. Miron (1), P. Blanchette (1), P. Groitl (2), F. Dallaire (1), J. G. Teodoro (1), S. Li (1), T. Dobner (2), P. Branton (1)(1) McGill University, Department of Biochemistry, Montreal, Canada (2) Heinrich-Pette-Institute, Hamburg, Germany

The human adenovirus type 5 (Ad5) E4orf4 product has been studied exten-sively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells. Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic back-ground. Such mutant viruses included those that express E4orf4 proteins con-taining various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluores-cent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.

*Supported by Canadian Institutes of Health Research – CIHR

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Intrinsic disorder in the amino terminus of human adenovirus type 5 E1B-55K and its related proteins

T. Sieber (1), R. Scholz (2), M. Spörner (3), H-R. Kalbitzer (3), T. Dobner (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) Swiss Federal Institute of Technology Zurich, Zurich, Switzerland (3) Institute for Biophysics and Physical Biochemistry, University of Regensburg, Germany

Early region 1B (E1B) of human subgroup C adenoviruses generates two major mRNA species of 22S and 13S with common 5’ and 3’ termini. Both 22S and 13S mRNAs are derived from a common mRNA precursor by alternative splicing. Translation of the 22S mRNA from adenovirus type 5 (Ad5) uses discrete, but over-lapping reading frames to produce two unrelated polypeptides of 176 amino acid residues (R) (E1B-19K) and 496R (E1B-55K). The 19K product is also translated from three alternatively spliced transcripts of 13S, 14S and 14.5S, which additionally direct the syn-thesis of three smaller 55K related proteins (156R, 94R and 83R) containing the amino-terminal 79 residues of E1B-55K. E1B-156R also shares an identical carboxy-terminus with 55K, while the 94R and 83R E1B polypeptides contain unique carboxy termini. The fact that four E1B proteins (E1B-55K, E1B-155R, E1B-94R and E1B-83R) share a common amino terminus indicates that this segment plays a key role in regulating the function of E1B-55K and its related proteins. Nevertheless, as yet very little is known about the function of this domain and the smaller E1B pro-teins in infection.

To gain further insight into the role the amino-terminal 79 amino acids for E1B function we performed NMR spectroscopy and other bio-physical methods with bacterially expressed E1B-93R to obtain high-resolution structural information on this protein region in solution. Our data show that E1B-93R, and, likely E1B-84R, are mostly unstruc-tured and fulfill the criteria of an

Ad5 E1B region and its gene products. Scale on top of the figure indi-cates nucleotide sequence position relative to the left end of the viral genome. The Ad5 E1B mRNAs and protein products predicted from DNA sequence and mapping studies have been illustrated. Included are also the relevant nucleotides for translation start and stop sites.

Ad5 E1B-93R is an intrinsically disordered protein. CD-spectrum of human Ad5 E1B-93R in a range of 195.5 to 260 nm. E1B-93R is mostly unfolded with a minor content of regular secondary structure. For comparison the theoretical spectra of pure α-helix, β-sheet and random coil were adapted to this figure.

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intrinsically disordered protein (IDP) with a high degree of dynamics and very little stable structure. In addition these findings, indicate that E1B-55K and E1B-156R contain an intrinsically disordered domain (IDD) in their amino-termini. IDPs comprise a rapidly growing family of proteins which, despite their lack of a well defined structure, often fulfill essential regulatory functions. The intrinsic lack of structure confers functional advantages on these proteins, allowing them to adopt multiple conformations and to bind to different binding partners. Consistent with this we found that the IDD mediates an interaction between the 55K related proteins and E1B-55K.

*Supported by ReFoMC program, University Regensburg

A novel 49K isoform of the adenovirus type 5 early region 1B 55K protein is sufficient to support viral replication

S. Schreiner (1), K. Kindsmüller (1), P. Groitl (1), T. Dobner (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

The adenovirus type 5 (Ad5) E1B-55K protein is a multifunctional regulator of cell-cycle independent virus replication that participates in many processes required for maximal virus production. As part of a study of 55K function, we generated a virus mutant (H5pm4133) carrying stop codons after the second and seventh codon of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the charac-teristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demon-strated that H5pm4133, and, significantly, wt Ad5 express low levels of a ~49K product, lacking the amino-terminal region of E1B-55K. Expression of the 49K iso-form was eliminated when the second and third downstream AUG at positions 64 and 75 of the E1B reading frame were converted to valine codons. Moreover, virus mutants producing the E1B-49K protein only, replicated to levels comparable to wt Ad5, while mutants lacking both E1B-55K and the 49K isoform were severely defec-tive for virus growth. These data demonstrate that the Ad5 E1B gene produces low levels of a novel 49K E1B protein species, which is generated by initiation of trans-lation at two downstream, in-frame AUGs of the E1B mRNA, and is sufficient to proceed through the productive infection in human tumor cell lines.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Daxx-mediated negative regulation of adenovirus replication is counteracted by the adenovirus E1B-55K protein

S. Schreiner (1), P. Wimmer (1), H. Sirma (1), R. Everett (2), P. Blanchette (3), T. Dobner (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) MRC Virology Unit, Glasgow, Scotland, UK (3) McGill University, Department of Biochemistry, Montreal, Canada

The death-associated protein Daxx found in PML nuclear bodies (PML-NBs), is involved in transcriptional regulation and cellular intrinsic immune defense

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against incoming viruses. We found that knock-down of Daxx using siRNA tech-niques in a non-transformed human hepatocyte cell line results in significantly increased in adenoviral (Ad) replication, including enhanced viral mRNA synthe-sis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is apparently counteracted by early protein E1B-55K, a multifunctional regulator of cell-cylce independent Ad replication. Here, we show that the Ad protein binds to Daxx and likely induces its degradation through a proteasome-dependent pathway. Remarkably, this process is independent of Ad E4orf6, known to promote the proteasomal degradation of cellular p53, Mre11 and DNA Ligase IV in combination with E1B-55K. These results illustrate the importance of PML-NBs and associated factors in virus growth restriction and suggest that two Ad early proteins (E1B-55K and E4orf3) synergistically antagonize the innate antiviral activities of PML-NBs to stimulate the viral replication program.

*Supported by Erich und Gertrud Roggenbuck-Stiftung

Posttranscriptional processing of HIV-1 mRNA

D. Hoffmann (1), M. Krepstakies (1), I. Hauber (1), B. Abel (1), H. Schaal (2), R.H. Kehlenbach (3), J. Hauber (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) University of Düsseldorf – Institute of Virology, Düsseldorf, Germany (3) University of Göttingen – Centre of Biochemistry and Molecular Cell Biology, Göttingen, Germany

During replication of the human immunodeficiency virus type 1 (HIV-1) the viral (+)RNA genome is reverse transcribed and integrated into the host cell genome. Transcription of this provirus by the cellular RNA polymerase II gener-ates a polycistronic pre-mRNA that contains at least four 5’ splice sites and eight 3’ splice sites that enable alternative splicing of more than 40 different mRNAs. These transcripts can be grouped into the three classes of RNA: The 2 kb-class is multiply-spliced and encodes the regulatory proteins Tat, Nef or Rev. The 4 kb-class is characterized by a single splicing event and encodes Env or the accessory proteins Vif, Vpr or Vpu. Finally, the 9-kb class consists of unspliced primary transcripts that either are used for Gag and Pol expression or act as genomes in the formation of progeny viruses.

The transport of the 4 kb class and 9 kb class of these RNAs is promoted by the retroviral Rev regulatory protein while, in contrast, nuclear export of the 2 kb class occurs independent of Rev. Rev is a nucleocytoplasmic shuttle protein that binds directly to the viral Rev response element (RRE) RNA sequence, which is present on all unspliced and singly-spliced HIV-1 transcripts (4 kb and 9 kb class). Subsequently, Rev interacts with multiple cellular factors that mediate the translocation of the Rev-bound viral RNA across the nuclear envelope.

The nucleocytoplasmic translocation of Rev-containing ribonucleoprotein (RNP) complexes is mediated by the general export receptor CRM1, which interacts in presence of RanGTP with a specific domain in Rev that has been termed the

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nuclear export signal (NES). However, efficient Rev-mediated nuclear RNA export also requires factors in addition to CRM1 and Ran. These apparently include, for example, eukaryotic initiation factor 5A (eIF-5A), the Src-associated protein in mitosis Sam68 and DEAD Box RNA Helicase DDX3.

In collaboration with Prof. Heiner Schaal (University of Düsseldorf) we investigated the translation efficiency of Env-encoding messages as well as the activation of alternatively used HIV-1 splice acceptors.

Translation of HIV-1 mRNAs generally follows the scanning mechanism. However, by using subgenomic env expression vectors, we found translation of glycoprotein from polycistronic mRNAs were inconsistent with leaky scanning. Close inspection of the leader sequences suggested expression of Env was modulated by an upstream open reading frame (uORF) and a minimal uORF consisting only of a start and stop codon that overlaps with the vpu start site. By altering the strength of upstream AUGs, the length of their coding region and the intercistronic distance, we demonstrate that each of these parameters influenced downstream translation. The minimal uORF is conserved throughout almost all HIV-1 subtypes, despite residing in a non-coding region of the genomic RNA. Mutating the translational start and stop codons of this uORF resulted in up to five fold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation

sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter env-expression.

Furthermore, the functional characterization of the mode of action of the guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) in exon 5 of the HIV-1 mRNA revealed, that this ESE fulfils a dual splicing regulatory function. By synergistically medi-ating exon recognition through its individual SR protein-binding sites and by conferring 3’ splice site selectivity within the splice site cluster preceding exon 5. As the GAR ESE-mediated activation of the upstream 3’ splice site cluster also is essential for the processing of intron-containing vpu/env-mRNAs, the GAR enhancer substantially contributes to the regulation of viral replication.

Distribution of splicing regulatory elements in the HIV-1 pre-mRNA. (A) Schematic depiction of the HIV-1 genome. (B) Distribution of 5’ splice sites (D1-D5) and 3’ splice sites (A1-A7) within the genomic HIV-1 RNA. The position of exon 5 is indicated (dark grey box). (C) Enlargement of the 3’ half of the pre-mRNA depicts the location of the known enhancer (green) and silencer elements (red) regulating viral pre-mRNA splicing. Exon 5 is flanked by 5’ splice site D4 and 3’ splice site A5 and contains the GAR ESE in the 5’ part (Cartoon by courtesy of H. Schaal).

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Another aspect of this project focussed on the nuclear uptake of the HIV-1 Rev RNA transport fac-tor. In particular we investigated in collaboration with PD Dr. Ralph Kehlenbach (University of Göttingen) the requirements for soluble and stationary components of the nucleocytoplasmic transport machinery for nuclear import of Rev in living cells. The respective data suggested that transportin is the major import receptor for HIV-1 Rev in vivo. Furthermore, nuclear import of Rev strongly depended on Nup358, a component of the cytoplasmic filaments of the nuclear pore complex. Moreover, Nup358 appears to act as a docking station for transportin-containing import complexes on their way into the nucleus, since nuclear import of other transportin-dependent sub-strates (e.g. hnRNP M) was also impaired in Nup358-depleted cells.

In addition to mediating nuclear export of HIV-1 mRNA, also Rev activities on the splicing, half-life and translation of these transcripts has been reported in the literature. In that respect an important and often overlooked feature of Rev is its ability to form homo-multimers. Various studies have demon-strated that Rev binds as a monomer to its high-affinity RRE binding site and subse-quently multimerizes on its RNA target. Although it has been unambiguously estab-lished that multimer formation is indeed required for Rev function, it is to date unclear which Rev activity depends on Rev multimer-formation. To address this question we also generated biologically inactive multimerization-deficient but shut-tling-competent Rev mutant proteins. By fusing these mutants to heterologous oli-gomerization structures we were able to demonstrate that formation of a Rev homo-dimer is sufficient to rescue the biological activity of these otherwise inactive Rev proteins. Furthermore, we were able to show that the cytoplamsic accumulation of RRE-containing retroviral transcripts depends on Rev multimer-formation. These findings support the notion that cellular cofactors in addition to CRM1 are required for Rev-regulated nucleocytoplasmic transport of HIV-1 RRE-containing mRNAs.

*Supported in part by Deutsche Forschungsgemeinschaft (DFG) and in part by Deutsche Krebshilfe

Transcription of the integrated proviral DNA (Provirus) produces full-length unspliced ~ 9 kb RNA, which is used for biosynthesis of Gag and Pol. In addition, this RNA species serves also as genome in the formation of progeny viruses. Splicing of the ~ 9 kb transcript results in RNAs of the single-spliced ~ 4 kb class, encoding Env, Vif, Vpu and Vpr, and of the multiple-spliced ~ 2 kb class, encoding Tat, Rev and Nef. Nuclear export and thereby translation of the ~ 9 kb and ~ 4 kb class of RNAs depends on the retroviral Rev trans-activator, which directly interacts with the Rev Response Element (not shown) present in these RNA species.

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Combating drug resistance in HIV-1 infection

P. Priyadarshini (1), P. Hartjen (2), I. Hauber (1), S. Balabanov (3), T. Brümmendorf (3), R. Hilgenfeld (4), C. Meier (5), M. Rarey (6), U. Holzgrabe (7), J. van Lunzen (1,2), J. Hauber (1) (1) Heinrich-Pette-Institute, Hamburg, Germany(2) University Medical Center Hamburg-Eppendorf – Infectious Diseases Unit, Hamburg, Germany (3) University Medical Center Hamburg-Eppendorf – Department of Oncology and Hematology, Hamburg, Germany (4) University of Lübeck – Institute for Biochemistry, Lübeck, Germany (5) University of Hamburg – Institute of Organic Chemistry, Hamburg, Germany (6) University of Hamburg – Center for Bioinformatics, Hamburg, Germany (7) University of Würzburg – Institute of Pharmacy and Food Chemistry, Würzburg, Germany

The introduction of highly active antiretroviral therapy (HAART) into clinical practice has pro-foundly improved the morbidity and mortality among HIV-1-infected patients. However, in a growing number of infected sub-jects long-term HAART is accom-panied by the occurrence of multi-drug-resistant (HAART-resistant) virus. Moreover, the transmission of multidrug-resistant HIV strains is becoming a growing problem among newly infected persons. In fact, recent studies have shown that 10-12% of viruses harbour mutations conferring resistance to one or more drugs or drug classes which are currently used in clini-cal practice to treat HIV-1 infec-tion. Thus, novel and advanced strategies have to be developed, particularly to combat the so far largely unmet medical problem of drug resistance in HIV-patients.

Previous studies revealed that eukaryotic initiation factor 5A (eIF-5A) is a critical cellular cofac-tor of the HIV-1 Rev regulatory protein, that primarily mediates

Pathway of eIF-5A biosynthesis. The unusual amino acid hypusine is a postranslational modification of eIF-5A and necessary for its activity. Hypusine is formed in two steps under catalysis of deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). DHS transfers an aminobutyl moiety from spermidine to a specific lysine residue in the eIF-5A precursor. The resulting intermediate is then hydroxylated by DOHH, generating active eIF-5A. CNI-1493 and JK8-2 are experimental small-molecular weight inhibitors of DHS and DOHH, respectively (Cartoon by courtesy of U. Holzgrabe).

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the translocation of incompletely-spliced and unspliced retroviral mRNAs across the nuclear envelope. This Rev cofactor is the only known cellular protein that contains the amino acid hypusine, a modification that is required for eIF-5A activity. The hypusine formation is a highly specific, spermidine-dependent post-translational reaction that is catalyzed by two enzymes. In the first step, the ami-nobutyl moiety of spermidine is transferred to the ε-NH2 group of a single lysine residue at position 50 within the human 154-amino-acid eIF-5A protein. This reaction is catalyzed by deoxyhypusine synthase (DHS). The resulting eIF-5A intermediate is then further modified by the metalloenzyme deoxyhypusine hydroxylase (DOHH), generating the active form of eIF-5A. Therefore, inhibition of the essential Rev cofactor eIF-5A by interference with hypusine-formation should provide a novel strategy to block the HIV-1 life cycle.

We previously identified the mul-tivalent guanylhydrazone CNI-1493 by high-throughput screen-ing assay to be a novel small-molecular weight inhibitor of the eIF-5A-modifying enzyme DHS. We demonstrated that this com-pound indeed efficiently inhibits Rev activity and thereby replica-tion of HIV-1, including the repli-cation of viruses that are other-wise fully resistant to classical HAART. Furthermore, in collabo-ration with Prof. Ulrike Holzgrabe (University of Würzburg) we characterized the antiretroviral activity of a novel DOHH inhibi-tors (e.g. JK8-2). In sum, these data provided the scientific rational (i.e. proof-of-principle) to establish an inter-institutional research network, including scientists from the Heinrich-Pette-Institute, the University Medical Center Eppendorf and the Universities of Hamburg and Lübeck, to investigate cellular eIF-5A function and to develop novel DHS/DOHH inhibitors. Since the hypusine-containing protein eIF-5A has also been identified to be a potential target for therapy of chronic myeloid leuke-mia (CML), various aspects of this interdisciplinary project also directly contrib-ute to the program area “Cellular Dysregulation” (for details see below). Thus, novel small molecular-weight DHS/DOHH inhibitors may allow to overcome drug-resistance in patients suffering from CML or HIV-1 infection.

During the initial project period the existing inhibitor lead-compounds were further characterized in vitro and in appropriate disease models. In particular, the antiretroviral activities of these compounds were investigated in various HIV-1 models. Furthermore, studies to analyze the potential influence of these com-pounds on the functional properties of T cells (i.e. HIV-specific CD4 and CD8 T

Inhibition of HIV-1 NL4/3 replication by the experimental DOHH inhibitor JK8-2. HIV-1 NL4/3-infected PM1 cells were cultured in the presence of the indicated concentrations of JK8-2, or DMSO as solvent control. Gag p24 antigen levels and cell viabilities (alamarBlue assays) were determined at day 6 and 9 post-infection. The percentage of inhibition of virus replication in the drug-treated cell culture, compared to the untreated controls, is shown. The analysis of the respective cell viabilities (not shown) revealed that the antiviral effect was not caused by deleterious effects of JK8-2 on the host cell.

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cell immune reponses) were initiated. Other aspects of this interdisciplinary project included the structural analysis of the hypusine-modifying enzymes, the computational molecular design of inhibitor analogues, the de novo inhibitor synthesis, and the detailled analysis of the cellular function of the hypusine-containing protein eIF-5A.

*Supported by Bundesministerium für Bildung und Forschung (BMBF) and in part by Deutsche Forschungsgemeinschaft (DFG)

Eradication of HIV-1 proviral DNA from patient cells

I. Hauber (1), I. Sarkar (2), J. Chemnitz (1), H. Hofmann-Sieber (1), C.-H. Nagel (1), A. Bunk (1), B. Weseloh (1), N. Beschorner (1), M. Krepstakies (1), A. Schambach (3), J. Chusainow (2), C. Baum (3), M. Manz (4), F. Buchholz (2), J. Hauber (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) Max-Planck-Institute for Molecular Cell Biology and Genetics, Dresden, Germany (3) Hannover Medical School, Hannover, Germany (4) Institute for Research in Biomedicine, Bellinzona, Switzerland

Human immunodeficiency virus type 1 (HIV-1), the causative agent of the aquired immunodeficiency syndrome (AIDS), integrates its proviral genome permanently into the chromosome of the host cell. Thus, HIV-1 establishes a

persistent lifelong infection. Current highly active antiretrovi-ral therapy (HAART), which is based primarily on virus enzyme inhibitors and molecules that inhibit virus attachement or virus-cell fusion does not target the integrated provirus and can there-fore not eliminate HIV-1 from the body.

In collaboration with Dr. Frank Buchholz (MPI-CBG, Dresden) we developed a novel strategy to combat and possibly cure HIV-1 infection through expression of in vitro taylored recombinases (Tre-recombinases). Tre-recombinases recognize asymmetric target sequences within the native Long Terminal Repeats (LTR) of the integrated provirus and excise the proviral HIV-1 DNA from the genome of infected cells.

(1) After entry into the cytoplasm, the RNA genome of retroviruses is reverse transcribed into DNA. Subsequently, this proviral DNA is integrated into the host genome and serves as template for the production of progeny viruses during the host cell’s entire life-span. (2) Reverse transcription results in sequence-identical long terminal repeats (LTR; indicated by grey boxes). Engineered LTR-specific recombinases (Tre-recombinase) recognize and recombine LTR sequences of the integrated proviral DNA. (3) Tre-recombinase activity results in the excision of the main body of the integrated proviral DNA, thereby eradicating retrovirus infection.

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In a proof-of-concept study we demonstrated that Tre-recom-binase quantitatively eliminates the provirus from a human cell culture infected with an HIV-1 Env-mutant in absence of cellular toxicity. To advance this novel therapy strategy towards clinical development we subsequently established an interdisciplinary project team.

Tre-recombinase therapy will most likely involve the introduc-tion of Tre-encoding sequences into the patient’s target cells by somatic gene transfer. We there-fore generated and analyzed in collaboration with Prof. Christopher Baum’s group (MHH) replication-incompetent and self-infactivating (SIN) lentiviral vec-tors of the newest generation.

A potential future Tre-therapy may be envisaged as follows: first, HIV-1 LTR sequences isolated from the blood of the infected subjects are determined and, sub-sequently, an adequate Tre-recombinase is generated in vitro by substrate linked protein evolu-tion. The respective Tre-encoding sequence is then be introduced ex vivo into the patient’s hematopoi-etic progenitor/stem cells (HSC) using lentiviral vectors. Following quality control, these genetically engineered HSC are then infused into the respective patient. It is expected, that the immune system of this patient will be functionally reconstituted over time, allowing the elimination of long-living latently-infected cells.

To further validate this therapy approach Tre-recombinase has been

The delivery of Tre-recombinase into infected patients most likely requires a gene therapy strategy. Current clinical protocols include, after G-CSF mobilization, the harvest of hematopoietic stem cells (HSC) by leukapheresis. Subsequently, ex vivo Tre-recombinase gene transfer using replication-incompetent and self-inactivating (SIN) lentiviral vectors and the subsequent autologous trans- plantation/infusion of the gene-modified cells would be performed. In consequence it is expected that HIV-resistant cells will increase in number. Remaining latently infected cells may now be targeted by the partially reconstituted immune system of the patient.

(A) A replication-incompetent and self-inactivating (SIN) lentiviral vector expressing Tre-recombinase and the eGFP reporter is depicted. (B) The gene encoding Tre-recombinase was delivered by the lentiviral SIN vector shown in panel A into PM1 cells. For control, PM1 cells were also infected with the parental SIN vector lacking Tre sequences. Subsequently, the respective cultures were infected with wildtype HIV-1 (BaL). HIV-1 replication was determined over a period of 10 weeks (w) in the Tre-expressing culture (Tre) and in the negative control culture (NC).

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successfully analyzed in cell cultures infected with fully replication-competent HIV-1 strain BaL. Furthermore, the target sequence specificity of Tre-recombinase has been evaluated in detail and potential Tre-induced toxicities were studied in various cel-lular systems.

To monitor Tre-activity in an infected organism we also initiated experiments in collaboration with Dr. Markus Manz (IRB, Bellinzona) employing Rag2-/-γc-/- mice. Transplantation of human cord blood-derived CD34+ HSC into newborns of these animals leads to de novo development of B, T, and dendritic cells; forma-tion of structured primary and secondary lymphoid organs; and production of functional immune responses. Consequently, these humanized animals can be infected with HIV-1. Therefore, we are currently transplanting Rag2-/-γc-/- animals with lentivirus-transduced human Tre-expressing HSC to evaluate in vivo Tre-recombinase activity.

*Supported by Bundesministerium für Bildung und Forschung (BMBF)

Rapid response kinetics to HLA-B57/58 Gag-restricted CTL epitopes is associated with slow disease progression in chronic HIV-1 infection

P. Hartjen (1), I. Stahmer (1), J. Schulze zur Wiesch (1), A. Thomssen (1), J.Hauber (2), J. van Lunzen (1,2)(1) University Medical Center Hamburg-Eppendorf – Infectious Diseases Unit, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

Correlations between a person’s HLA composition and the ability of cytotoxic T lymphocytes (CTL) to control HIV-1 replication are well documented. Especially B27 and B57/58 restricted CTL responses directed against Gag-derived epitopes are associated with slow disease progression. We were inter-ested in defining the underlying mechanisms that may account for the benefi-cial role of these particular immune responses. Therefore, we assessed the response kinetics of CTL from HIV infected subjects with slow disease progres-sion in an IFN-γ-ELISPOT assay and phenotypically characterized immunodo-minant CTL by tetramer staining.

So far, cyropreserved PBL derived from 21 HIV-1 infected subjects were analyzed with different characteristics in disease progression. Patients with non-progres-sive disease or slow progression and good viral control in the absence of HAART were compared to a HLA-matched control group with progressive disease with or without HAART according to current treatment guidelines. Patients were selected on the basis of their HLA haplotype to ensure the same frequencies of “beneficial” B57/58 and B27 haplotypes in both groups. Multiple samples were analyzed lon-gitudinally (range 1-6 years) using a specialized IFN-γ-ELISPOT assay using optimal B clade consensus peptides for HLA-A and -B haplotypes derived from Gag, Pol, Nef, and/or Env regions.

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HLA-B57/58 and B27 haplotypes were evenly distributed in LTNP and controls (8/10 vs. 8/11). The B57-and B58-restricted CTL-responses against Gag-epitopes were detected to be the immunodominant response in these patients. Less frequent-ly, B44-Gag, B44–Env and B27-Gag restricted responses were detected in descending order. These strong CTL responses were detected in LTNP as early as 30 min after pep-tide exposure in vitro and gradually increased thereafter. This rapid response pattern was conserved in LTNP for more than 2 years and lasted up to 6 years of follow up. In contrast, most of the HLA-A-restricted responses were only detectable after 24 hours of in vitro stimulation, suggesting a compara-tively delayed response kinetic for non-B57/58 restricted epitopes in the control group. Interestingly, HLA-matched patients with pro-gressive disease displayed consider-ably slower and weaker Gag-restricted responses, even in patients with B57/58 haplotype.

We further characterized immuno-dominant CTL by tetramer stain-ing. In order to distinguish between the differentiated subsets “early”, “intermediate” and “late” (CD28+CD27+, CD28-CD27+ and CD28-CD27-, respectively) we determine the expression level of the costimulatory receptors CD27 and CD28 in tetramer-positive CTL. This clearly revealed an enrichment of CTL presenting “early” memory pheno-type in individuals with slow disease progression, which is associated with controlled acute viral infection (e.g. EBV, CMV, chronic HIV). Interestingly, this phenotype shifted towards “intermediate” and “late” phenotypes resembling uncontrolled chronic infection in patients with progressive disease (e.g. chronic HIV/HCV).

We conclude, that the dominance of B57/58 haplotypes in slowly progressing HIV-1 infection is not only explained by the breadth and magnitude of CTL-responses but also by the rapid response kinetics of specific CTL in patients with slow disease pro-gression. These Gag-specific CTL may help preventing establishment of HIV-1 latency via targeting the virus life cycle very early at the pre-integration level.

CD8+ T cells are schematically assigned to distinct functional subsets. A hypothetical model of linear CD8+ T-cell differentiation is depicted. Four main phenotypic subsets as well as their respective enrichment into memory virus-specific CD8+ T cells are presented. L and H indicates low expression and high expression, respec-tively. Phenotypes in brackets represent intermediate or minor populations. Cartoon adapted from Appay et al. 2002 Nature Medicine 8:379-85.

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Determinants of successful antiviral treatment of concomitant acute HCV/HIV coinfection.

J. Schulze zur Wiesch (1), D. Pieper (2), A. Thomssen(1), P. Hartjen (1), J. Hauber (2), J. van Lunzen (1,2) (1) University Medical Center Hamburg-Eppendorf – Infectious Diseases Unit, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

HIV and HCV share similar routes of infection and chronic HCV coinfection with all its complications has become a major health threat to HIV-infected individuals worldwide. Lately, outbreaks of sexually transmitted acute HCV infection in chroni-cally HIV-infected patients have been reported from different parts of Europe. So far only limited data exist concerning the clinical outcome and the immunologic responses which are primed during simultaneously acquired acute HCV/HIV co-infection. In a pilot study we conducted a detailed clinical and immunologic analysis of three individuals following concomitant acute HCV/primary HIV infection. Additionally to longitudinal clinical parameters, virus-specific CD8+ and CD4+ T cell responses were assessed using ELISPOT, standard proliferative (CSFE) and further in vitro CD4+ T cell assays. In all patients anti-HCV treatment was started and HIV antiretroviral therapy (ART) was co-administered early during primary infection. HCV viremia was cleared under therapy with pegylated IFN-alpha in all three cases. In two patients HIV replication was contained even after ART was interrupted, which was associated with strong HIV-specific CD8+ and CD4+ T cell responses. In these two patients multi-specific HCV CD4+ responses could also be detected. No HCV spe-

cific CD4+ responses were detected in the third patient who also had the lowest CD4+ nadir during PHI (<200 cells/l). Our data indicate that HCV antiviral therapy should be considered early in the setting of concomitant acute HCV/HIV co-infection and that a sustained viro-logical response is possible even during primary HIV infection. Clearly, larger prospective trials have to determine the best anti-HCV treatment regimen and whether early adjuvant antiretroviral treat-ment is needed to achieve the favo-rable clinical and immunological outcome described here. A better understanding of immune respons-es following acquisition of HCV may shed insight into both, HCV pathogenesis in HIV-infected indi-viduals and prospect for immuno-therapies in this population.

Immunologic control of HCV pathogenesis. In a normal HCV mono-infection, HCV-specific CD4 and CD8 T cells are mobilized to contain infection. In case infection is not cleared, such T cells contribute to antiviral, proinflammatory, and regulatory effects. There is emerging evidence that the high viral loads of HCV during coinfection with HIV are associated with profound defects in HCV-specific CD4 and also CD8 T cell responses. Moreover, some preliminary experimental data suggest that the cytokine environment in the liver is altered, which could affect disease progression independently from any influence of virus load (graphic courtesy of A. Kim, Boston).

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Crossing the Species Barrier: Role of Env-Receptor Interactions in Gammaretrovirus Infections

U. Bergholz (1), M. Ziegler (1), K. Stieler (2), A.D. Miller (3), M. Eiden (4), N. Fischer (2), V. Prassolov (5), C. Stocking (1), (1) Heinrich-Pette-Institute, Germany (2) University Medical Center Hamburg-Eppendorf, Hamburg, Germany (3) Fred Hutchison Cancer Center, Seattle, WA USA (4) National Institute of Health, Bethesda, MD, USA (5) Engelhardt-Institute of Molecular Biology, Moscow, Russia

The gammaretroviruses compromise a genus of exogenous and endogenous viruses that are widely spread in vertebrates. Analysis of endogenous retrovirus (ERVs) in the genomes of humans, mice, and other species indicates a longstanding associa-tion, probably dating back several hundred million years, during which retroviruses have repeatedly colonized host genomes. Phylogenetic studies of class I (gamma and epsilon) and class II (alpha, beta, delta and lenti) ERVs suggest that horizontal trans-fer of infectious virus between vertebrate classes occurs only rarely, although several important examples have occurred. In addition to the well-known jumping of a lentivirus from chimpanzees (SIVcpz) to humans (HIV-1), a recent report has dem-onstrated the first bonafide human infection with a xenotropic MLV-related gam-maretrovirus (XMRV). Using retroviral pseudotype assays and receptor binding assays, we have demonstrate that XMRV possesses a wide host range and efficiently infects feral mouse cells, as well as established human cell lines and primary cells. Interference assays confirm the classification of XMRV as a xenotropic MLV-like virus, which uses the transmembrane protein Xpr1 as a receptor. Interestingly, XMRV preferentially uses the human versus the murine Xpr1 variant, arguing that XMRV already persists for a longer period in the human population.

Another example of cross-species transfer is the recent characterization of a Koala gammaretrovirus (KoLV), which is currently "invading" the host genome. This virus is closely related to the gibbon ape leukemia virus (GALV), although the vector responsible for transmission of the virus between these two species with distinct habitats has not been conclusively identified. We have recently iso-lated an ERV from Mus cervicolor, which we have dubbed McERV. McERV is closely related to the GALV and KoLV isolates and thus may share a common ancestor (Fig. X). Host and tissue spectrum analysis has shown that in contrast to most other gammaretrovirus isolates that use ubiquitously expressed membrane transporters as a receptor, McERV uses the myelin protein plasmolipin (PLLP) as a cellular receptor. PLLP expression pattern is restricted to cells of nervous and secretory systems (e.g. brain, spinal cord, kidney, and lungs) and thus retroviral vectors pseudotyped with McERV show tissue expressions. We are currently screening the mouse genome for other uncharacterized ERVs that may shed light on the evolution and spread of gammaretrovirus, but also provide attractive tools for cellular and molecular biology, as well gene therapy.

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Requirement of Activation for Hepatitis B Virus Infection.

M. Bruns (1), C. Maenz (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

Although in vitro models of human hepatitis B virus replication are established, so far none could approximate infection efficiency as expected from in vivo observations. Susceptibility for HBV infections has only been reported for pri-mary hepatocytes of human, chimpanzee or Tupaia belangeri and the cell line HepaRG. We showed that the insusceptible human hepatoma cell line HepG2 can be infected, when the virus was beforehand activated by passage over whole duck liver cell cultures. That suggests an activation step to be performed by specialized liver cells.

*Supported by Deutsche Forschungsgemeinschaft

Enzymatic treatment of duck hepatitis B virus: topology of the surface proteins for virions and noninfectious subviral particles

M. Bruns (1), C. Franke (1), U. Matschl (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles’ inte-rior or exterior. After separating virions from subviral particles (SVPs), we com-pared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: first, preS of intact virions was enzy-matically digested with chymotrypsin, whereas in SVPs only half of preS was accessible; second, phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions; third, iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

*Supported by the Deutsche Forschungsgemeinschaft

Entry of Duck Hepatitis B Virus into Primary Duck Liver and Kidney Cells after Uncovery of a Fusogenic Region within the Large Surface Protein

C. Maenz (1), S.F. Chang (1,2), A. Iwanski (1), M. Bruns (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Biochemical Engineering Center, Chutung Hsinchu, Taiwan

Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus it has been shown that the preS domain of L binds to carboxy-

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peptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless only hepatocytes become infected. It so far remained vague, which viral features procure host range specificity and organotropicity. Using chymo-trypsin to treat duck hepatitis B virus we addressed the question, whether a puta-tive fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the requirements for the host range of the virus. The addition of the enzyme to virions resulted in an increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory for infection. A noninfectious deletion mutant, devoid of the binding region for carboxypeptidase D, could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although due to the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, the host range specificity was not affected as shown by the inability of viral replication in different hepatoma cell-lines as well as primary chicken hepatocytes. Instead, the organotropicity could be reduced, which was demonstrated by infection of primary duck kidney cells.

*Supported by Deutsche Forschungsgemeinschaft

Elucidation of the SV40-induced DNA damage signaling cascades and their components utilized by SV40

G. Rohaly (1), K. Korf (1), S. Dehde (1), A. Mena-Nunez (1), H. Will (1), I. Dornreiter (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

Simian Virus 40 (SV40) infection of permissive wildtype p53 (wtp53) CV-1 cells activates the phosphoinositide 3-kinase-related kinase (PIKK) ATM (Ataxia telangiectasia mutated) leading to ATM-catalyzed phosphorylation of cellular down-stream substrates Chk2, H2AX, SMC1, p53 and viral T-Ag. Inactivation of ATM by dominant-negative effects shortly after viral infection prevented phos-phorylation of the aforementioned down-stream substrates, and unexpectedly impaired T-Ag expression. Thus, data strongly indicate that SV40 utilizes ATM to transactivate its early region, which encodes for the replication/transcription fac-tor T-Ag. Accordingly, in SV40-infected, ATMkd expressing cells viral amplifica-tion and T-Ag-mediated transactivation of the late region, which encodes for the capsid proteins, were completely abrogated. Another PIK kinase, which is acti-vated upon viral infection, is ATR (ATM and Rad3-related protein). Inactivation of ATR by Dox-induced expression of ectopic ATRkd shortly after viral infection did not interfere with T-Ag expression and ATM activation, as corroborated by the appearance of phosphorylated Chk2, H2AX, SMC1, p53 and T-Ag. However, SV40-infected TO-CV-1 cells expressing dominant-negative ATRkd failed to acti-vate the transcriptional activity of Δp53, an activity that leads to up-regulation of Cdk-inhibitor p21 and concomitantly inactivation of the S-phase promoting cyclin A-Cdk2 complex. Furthermore, our data show that viral amplification was significantly reduced (90%) in ATRkd expressing cells, indicating that ATR-Δp53-p21-mediated inactivation of cyclin A-Cdk2 is essential for maximal viral ampli-

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fication. In SV40-infected CV-1 cells, about ten active viral replication centers are detectable 24 hours post infection, which can be identified by EdU-incorporating large T-Ag foci. In contrast, in SV40-infected, TO-CV-1-Flag-ATRkd cells forma-tion of active viral replication centers was considerably impaired, despite cellular replication, as evidenced by cellular EdU-incorporation and EdU-negative T-Ag foci. In addition, inactivation of the ATR down-stream target Δp53 by expressing mutΔp53 or preventing up-regulation of Δp53-transactivated p21 via expression of shRNAp21 also impaired the formation of active viral replication centers, although to a lesser extend. Thus, the ATR-Δp53-21-mediated inactivation of cyclin A-Cdk2 is indispensable for facilitating viral amplification. Taken together, our data demonstrate that the ATM- and the ATR-mediated DNA damage response signaling pathways are purposefully activated in SV40-infected permis-sive cells, to enable adaptation of the host cell’s milieu for optimal viral progeny.

*Supported by Deutsche Forschungsgemeinschaft

Role of the host’s p53 status in SV40 life cycle

K. Korf I (1), G. Rohaly (1), S. Dehde (1), H. Will (1), I. Dornreiter (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

Although the transcriptional activity of p53 is inactivated in SV40-infected cells, we propose that p53 plays a role in the SV40 life cycle. The assumption is sup-ported by the finding that for the human adeno-associated virus type 2 (AAV2) it was shown that intact p53 is required to prevent premature death of the host cell and thus contributes to enhanced viral progeny. Therefore, we tested whether the p53 status of the host influences the infection process and accordingly viral amplification. Evaluation of viral replication efficiency in SV40-infected wtp53 CV-1 and mutp53-175D expressing wtp53 TO-CV-1 cells revealed that viral amplification was, in comparison to the parental cells, significantly reduced

Formation of active SV40 replication centers depends on functional ATR.

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(70%) in TO-CV-1-Flag-mutp53 cells. Analysis of the cell cycle distribution did not display any differences between wtp53 and mutp53 expressing CV-1 cells. In either cell lines, the ATM-Chk2- and the ATR-Δp53-p21-mediated DNA damage pathways were fully activated, leading to attenuation of S-phase progression, thereby providing an optimal environment for viral amplification. In addition, expression of the viral proteins VP1 and T-Ag as well as ATM-catalyzed phospho-rylation of T-Ag were not impaired. Thus, at first sight no obvious reason was found, which would explain the negative impact of mutp53 on viral progeny. Since T-Ag interacts with wtp53 and the tumor suppressor is recruited into viral replication centers one possible explanation is that the p53 hot spot mutant 175D interferes with the viral replication process. Indeed, complex formation of mutp53 with T-Ag as well as recruitment of mutp53 to viral replication centers, which displayed diminished replication activity, was observed. Data strongly indicate that the p53 status of the host cell plays an important part in SV40 DNA replication. However, the molecular mechanisms involving transcriptional inac-tive wtp53 in viral amplification remain to be elucidated.

Elucidation of the molecular mechanisms that underlie SV40-induced polyploidization

G. Rohaly (1), S. Dehde (1), K. Korf I (1), A. Mena-Nunez (1), H. Will (1), I. Dornreiter (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

The current model of origin licensing proposes a sequential assembly of pro-teins to form the pre-replication complex (pre-RC) and describes the basic mechanisms responsible for preventing re-initiation of DNA synthesis within a single cell cycle. In the first step, Cdc6, Cdt1 and cyclin E collaborate with the origin recognition complex to load the replication helicase (Mcm2-7) into pre-RCs at replication origins. In the second step, two protein kinases, Cdc7-Dbf4 and the cyclin E-Cdk2, convert pre-RCs into bidirectional replisomes at each origin. Re-initiation at replication origins within a cell cycle is prevented by the inactivation of pre-replication components in S- and G2-phase. The Cdks play a central role in establishing a block to re-replication through phosphorylation of each of the components. At present several proteins of the mammalian pre-RC have been described to be phosphorylated by cyclin A-Cdk and as a result degraded or inactivated. First, it was demonstrated that cyclin A-Cdk2-dependent Cdt1 phosphorylation in S-phase induces its degradation. Second, the licensing factor Cdc6 remains associated with chromatin throughout S-phase, but is inactivated by cyclin A-Cdk2 catalyzed phosphorylation. Third, cyclin A-Cdk2 phosphorylation decreases the helicase activity of the Mcm com-plex, reduces the complex’s binding to chromatin and may be at least partially responsible for the gradual release of chromatin-bound Mcm proteins. Forth, cyclin A-Cdk2 phosphorylation of origin bound DNA polymerase α-primase complex (Pol α) immediately after origin firing prevents re-loading of the replicase and accordingly re-initiation of bidirectional DNA replication. Last,

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cyclin A-Cdk1 phosphorylation of the tetrameric Pol α complex prevents overall loading of the replicase onto chromatin.

In SV40-infected permissive wtp53 cells, the continued replication of host cell chromosomal DNA beyond 4N implies alteration of the normal controls acting to limit DNA replication to once per cell cycle. Considering that cellular mech-anisms blocking re-replication of DNA act by cyclin A-dependent phopshoryla-tion of the aforementioned replication factors it is feasible to suggest that dys-regulation of replication control in SV40-infected cells is altered at the Cdk activity and cyclin expression level. Indeed, our results demonstrated that SV40 infection leads to inhibition of cyclin A-Cdk activity and accordingly S-phase arrest, which is accomplished by the ATR-Δp53-p21-mediated DNA damage pathway. Moreover, in SV40 infected cells degradation of the licensing factor Cdt1 was not observed; instead, the factor remained chromatin associated until the cells displayed a cytopathic effect. Another factor, which is an essential com-ponent of the origin-licensing complex and in concert with Cdk2 converts the pre-RC into an active replication complex is cyclin E. In normal cycling cells cyclin E is loaded onto chromatin during G1-phase and promotes loading of the Mcm helicase into pre-RCs. Accordingly, expression of cyclin E is tightly regulated so that peak levels occur at the G1/S boundary followed by a decline and loss of expression during S-phase. Thus, degradation of cyclin E prevents re-loading of the Mcm helicase and assembly of pre-replication complexes in S-phase. However, in SV40-infected permissive cells cell cycle-dependent turn-over of cyclin E is impaired due to sequestration of the Cul4 E3 ubiquitin ligase by T-Ag, leading to accumulation of cyclin E as well as high cyclin E-Cdk2 activity. We found that in SV40-infected cells proteasome-resistant cyclin E remains chromatin associated throughout S-phase, and together with Cdt1 facilitates loading of the Mcm helicase onto chromatin, thereby enabling re-licensing of RCs. In addition, cyclin E-Cdk2 activity is essential to promote loading of replication factors Cdc45 and Pol α into the licensed RCs. Since SV40-infected cells display high cyclin E-Cdk2 activity and are devoid of the replication antagonist cyclin A-Cdk2, loading of Cdc45 and Pol α onto the re-licensed origins of replication is permitted. Taken together, our investigations demonstrate that SV40 overrides DNA replication control through Cdk/cyclin-deregulation, which is facilitated by the ATR-Δp53-p21- and the ATM-mediated DNA damage response pathways.

Inhibition of duck hepatitis B virus infection of liver cells by combi-ned treatment with viral e antigen and carbohydrates

C. Maenz (1), C. Loscher (1), A. Iwanski (1), M. Bruns (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

The e antigen (eAg) of duck hepatitis B virus (DHBV) is a glycosylated secretory protein with a currently unknown function. We concentrated this antigen from the supernatants of persistently infected primary duck liver cell cultures by ammonium sulphate precipitation, adsorption chromatography over concanava-

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lin A Sepharose, preparative isoe-lectric focusing and molecular sieve chromatography. The com-bined treatment of duck liver cells with DHBV eAg (DHBe) concen-trate and alpha-methyl-d-man-nopyranoside strongly inhibited DHBV replication at de novo infection. When DHBe was added to non-infected primary duck liver cells, it was found to be associated with liver sinusoidal endothelial cells. This binding could be inhib-ited by the addition of alpha-methyl-d-mannopyranoside and other sugar molecules. The inhibi-tory effect of DHBe on infection could play a role in maintaining viral persistence.

*Supported by Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung

Solution structure of stem-loop alpha of the hepatitis B virus post-transcriptional regulatory element

M. Schwalbe (1), O. Ohlenschläger (1), A. Marchanka (1), R. Ramachandran (1), S. Häfner (1), T. Heise (2), M. Görlach (1)(1) Leibniz-Institut für Altersforschung/Fritz-Lipmann-Institut, Jena, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regu-latory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral tran-scripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepadnaviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα.

*Supported by Deutsche Forschungsgemeinschaft

Purification of DHBe. (a) Elution profile for DHBe over a Concanavalin A-Sepharose column; (b) Isoelectric focusing of DHBe within a pH gradient of 3.5-10; (c) Molecular sieve chromatography of DHBe in Sephadex G-75; (d) Analysis of purfication steps by electrophoresis: staining with Coomassie blue (left) or Western blotting with anti-DHBc antiserum (right).

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Analysis of Duck Hepatitis B Virus core protein phosphorylation and dephosphorylation

C. Maenz (1), M. Bruns (1), H. Will (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

The core proteins of hepatitis B viruses are phosphorylated at serin/threonine sites during virus entry, shortly after or concomitantly during core protein neosynthesis, and are dephosphorylated during viral DNA maturation short-ly after core assembly. It is neither known which of the many ser/thr sites are (de)modified at which stage of infection, nor which enzymes are involved, nor whether the dynamic on/off of phospho groups is functionally relevant. In order to address some of these questions we have overexpressed two known cellular phosphatases (phosphatase 2B (calcineurin) and phosphatase 2A), which we considered candidates potentially involved in core dephosphoryla-tion, and tested their effects. Both enzymes induced hypophosphorylation of the core protein compared to non-transfected cells but had no (with cal-cineurin) or only little effect (with PP2A) on steady state levels of viral pro-tein expression, viral DNA synthesis, formation and secretion of viral parti-cles as well as on infectivity of the secreted viral particles for primary duck liver cells. Studies with several duck hepatitis B virus (DHBV) core protein phosphosite specific antibodies which we produced indicated differential effects of each enzyme for specific DHBV core phosphosite. Overexpression of a dominant negative mutant of PP2A increased the level of intrahepatic DHBV core protein and slightly increased viral particle secretion. Taken

Hypophosphorylation of DHBc by Calcineurin. Detection of DHBV core protein and calcineurin in transfected hepatoma cells by indirect immunofluorescence staining (left panel) and calcineurin-mediated dephosphorylation of DHBc but not of the envelope protein PreS as evidenced by immunoblotting (right panel).

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together, these data argue against an essential role of phosphorylation at any stage of DHBV core phosphorylation and suggest a stimulatory role of phosporylation of unknown cellular and/or viral proteins for DHBV progeny production.

*Supported by Deutsche Forschungsgemeinschaft

Transactivation activity of the X and PreS protein of duck hepatitis B virus are dispensible for DHBV infection in vitro and in vivo

C. Kranig (1), K. Dallmeier (2), C. Maenz (1), M. Bruns (1), H. Will (1)(1) Heinrich-Pette Institute, Hamburg, Germany (2) Institute of Virology, University Freiburg, Germany

Both human and duck hepatitis B viruses (HBV and DHBV) code for two pro-teins with transcriptional transactivation activity, the X proteins and the large envelope proteins preS in phosphorylated forms. HBV mutants lacking both activities cannot produce virions in hepatoma cells, whereas HBV deficient in only one of both are viable. We have investigated whether the same applied to DHBV. Viral DHBV DNA genomes unable to produce DHBx and phospho-preS or both were created by mutagenesis. These mutant genomes were functionally tested both in chicken hepatoma cells (LMH) by transfection, in primary duck hepatocytes as well as in ducklings. All three mutant DHBV genomes were as efficient in viral protein synthesis, viral replication and production of progeny as the wildtype genome in LMH cells. Unexpectedly, there was also no detectable difference in these parameters between the mutants and wildtype DHBV in infec-tivity and production of progeny when analysed in primary duck hepatocytes and in ducklings. These data indicate that the transactivation activity of DHBx and phospho-preS of DHBV are dispensible for the infectious life cycle of DHBV in vitro and in vivo.

*Supported by the Bundesministerium für Bildung und Forschung

Host-range and pathogenicity of hepatitis B viruses

H.-J. Netter (1), S.-F. Chang (2), M. Bruns (3)(1) Monash University, Department of Microbiology, Australia (2) Industrial Technology Research Institute, Chutung Hsinchu, Taiwan (3) Heinrich-Pette-Institute, Hamburg, Germany

Hepatitis B viruses are small enveloped DNA viruses referred to as Hepadnaviridae that cause transient or persistent (chronic) infections of the liver. This family is divided into two genera, orthohepdnavirus and avihepad-navirus, which infect mammals or birds as natural hosts, respectively. They possess a narrow host range determined by the initial steps of viral attach-ment and entry. Hepatitis B virus is the focus of biomedical research owing to its medical significance. Approximately 2 billion people have serological evidence of hepatitis B, and of these approximately 350 million people have

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chronic infections (World Health Organisation, Fact Sheet WHO/204, October 2000). Depending on viral and host factors, the outcomes of infec-tion with hepatitis B virus vary between are acute hepatitis, mild or severe chronic hepatitis or cirrhosis. Chronic infections are associated with an increased risk for the development of hepatocellular carcinoma. We have reviewed specific aspects of the host range and pathogenicity of hepatitis B viruses which became recently apparent.

Polyomavirus-encoded miRNAs

N. Fischer (1), N. Walz (2), T. Christalla (2), T. Günther (2), U. Tessmer (2), A. Grundhoff (2)(1) University Medical Center Hamburg-Eppendorf, Institute of Med. Microbiology, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

Previously, we have developed a computational algorithm (VMir) for the ab initio prediction of viral miRNAs, and sucessfully applied the method to iden-tify miRNAs in several viruses, including the Polyomavirus SV40. SV40 encodes a single pre-miRNA hairpin, which produces two mature miRNAs late in viral infection. These miRNAs targets the early transcripts which encode large T-antigen (LT-Ag), thus preventing accumulation of superfluous amounts of

A miRNA encoded by Merkel Cell Polyomavirus (MCPyV). (A) VMir Prediction of putative miRNAs encoded by MCPyV. miRNA candidates are shown by genomic position (x-axis) and score (y-axis). The experimentally verified, high scoring candidate is shown in red and marked with an arrow. Predictions with a score of less than 115 (dotted line) are deemed insignificant. (B) Experimental validation (small RNA northern blot) of the miRNA candidate predicted by VMir in 293T (left) and 293 (right) cells transfected with two different constructs (MCPyV I + II) which express subgenomic regions harboring the miRNA candidate. The position of the mature miRNA is marked with an arrow. (C) Predicted structure of the MCPyV miRNA precursor stem loop. The position of the mature miRNA is shown in red.

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the highly immunogenic LT-Ag protein. The pre-miRNA hairpin is located just distal of the open reading frame encoding the major capsid antigen. It is fully complementary to the LT-Ag transcripts which are being transcribed from the other strand, and thus exerts its negative regulatory effect via cleavage of these transcripts in an siRNA like manner. As predicted by VMir (and according to subsequent experimental confirmation), the closely related polyomaviruses JVC and BKV also encode a single pre-miRNA. These pre-miRNAs are located at a similar genomic location as the SV40 miRNA, but are not related in sequence. Nevertheless, as they are located antisense to the LT-Ag gene, they retain their ability to negatively regulate LT-Ag transcripts via cleavage.

We have analyzed the recently discovered Merkel cell polyomavirus (MCPyV), a novel polyomavirus which is thought to play a causative role in the pathogenesis of Merkel cell carcinoma (MCC). A high scoring miRNA candidate predicted by VMir was experimentally tested and found to encode a bona fide miRNA.

Interestingly, the MCPyV-encoded miRNA is encoded opposite to sequences forming the proximal portion of the LT-Ag ORF, at a fundamentally different genomic position than the miRNAs of SV40, JCV and BKV miRNAs. Phylogenetic analysis shows that MCPyV is more closely related to the Mouse Polyomavirus (MuPyV) branch of the Polyoma-virus family than to SV40, BKV and JCV. Indeed, VMir analysis suggests that MuPyV, Hamster Polyomavirus (HPyV) and

Polyomavirus-encoded miRNAs. (A) Phylogenetic tree of the Polyomavirus family. The human members of the family are shaded grey. (B) Alignment of selected Polyomavirus genomes and position of experimentally verified (red) or VMir-predicted (blue) miRNAs, indicated by block arrows. Sequences encoding small, middle and large T-Ag or capsid antigens (VP1, VP2 and VP3) are shown as yellow or green block arrows, respectively. Aligned genomic regions are symbolized by the intermittent black line underneath. Gaps in the line indicate the absence of sequences present in other genomes. (C) Alignment of sequences of mature 5p and/or 3p miRNAs encoded by SV40, BKV, JCV, Merkel Cell Polyomavirus (MCPyV) and Mouse Polyomavirus (MoPyV), showing the absence of significant sequence conservation. The SV40, BKV and JCV pre-miRNA hairpins pro-duce mature miRNA molecules from both arms.

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the lymphotropic Polyomavirus (LPV) all harbor miRNAs at a similar location as MCPyV.

While Polyomavirus-encoded miRNAs thus are overall neither related in sequence nor genomic location, they are nevertheless predicted to retain complementarity to LT-Ag transcripts, suggesting the negative regulation of LT-Ag is functionally important in vivo.

In addition to the experimental validation of HPyV and LPV miRNAs, we are currrently investigating the molecular mechanisms governing polyomavirus miRNA expression as well as the role of the MCPyV miRNA in the viral lifecycle and its possible contribution to cellular transformation by MCPyV.

*Supported by Bundesministerium für Gesundheit (BMG)

A Global Analysis of Evolutionary Conservation Among Known and Predicted Gammaherpesvirus microRNAs

N. Walz (1), T. Christalla (2), U. Tessmer (2), A. Grundhoff (2)(1) University Medical Center Hamburg-Eppendorf, Institute of Med. Microbiology, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

MicroRNAs (miRNAs) are small, non-coding RNAs which post-transcription-ally regulate mRNA expression. The current release of the miRNA registry lists a total of 140 viral miRNAs. Strikingly, the vast majority (137) of these are encoded by members of the herpesvirus family. Whereas the propensity to

Conservation of EBV and rLCV miRNAs. (A) Schematic depiction of the genomic location of EBV- and rLCV-miRNAs and their conservation. Open reading frames are shown as block arrows, the location of pre-miRNAs is indicated by triangles underneath or above the EBV and rLCV backbones. pre-miRNA hairpins are depicted in higher detail in the center: Light gray lollipops drawn proximal to the backbone symbolize non-conserved pre-miRNAs, grey or black lollipops depict conserved hairpins. Solid or dotted lines connect homologous hairpins and indicate whether conservation includes the seed region of mature miRNAs or is limited to the pre-miRNA (dotted line). (B) Experimental confirmation of novel rLCV and EBV miRNAs by small RNA northern blotting.

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encode miRNAs thus appears to be a conserved feature of herpesviruses, the miRNAs themselves show little conservation: with the exception of 7 miRNAs known to be shared between Epstein-Barr Virus (EBV) and its close relative Rhesus Lymphocryptovirus (rLCV), all other herpesvirus miRNAs appear unrelated in sequence. We have recently employed VMir, a computational ab initio prediction method, to identify miRNAs in several viral genomes, including 12 miRNAs encoded by Kaposi's sarcoma-associated herpesvirus and 23 miRNAs encoded by EBV. We have modified the algorithm to allow consideration of evolutionary conservation and conducted a global analysis of all fully sequenced gammaherpesvirus genomes currently listed in the NCBI database.

Our analysis confirmed the conservation of miRNAs between EBV and rLCV, but predicted additional conserved miRNAs; indeed, subsequent experimental validation confirmed the existence of 2 novel EBV- and 16 novel rLCV-encoded miRNAs, with a total of 14 miRNAs sharing recognizable sequence homology. In contrast, with the exception of viral genomes with a very high degree of overall sequence conservation (i.e., different viral strains such as KSHV type M and P, as well as the closely related rhadinoviruses Macaca Fuscata Rhadinovirus and

Conservation of Gammaherpesvirus miRNAs. (A) All complete gammaherpesvirus genomes deposited in the Refseq database were blasted against each other. The upper panel the percentage of overall sequence identity between individually aligned genomes. The lower panel shows the number of confirmed pre-miRNAs or pre-miRNAs predicted by VMir which were found to be conserved between genome pairs (Self-aligned genomes show the total number of predicted pre-miRNAs). (B) VMir Prediction of Equid herpesvirus 2 (EqdHV2), Ovine herpesvirus 2 (OvHV2), Alcelaphine herpesvirus 1 (AlHV1) miRNAs. These viruses are predicted to express large numbers of miRNAs unrelated in sequence, but encoded at similiar genomic locations.

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Rhesus Rhadinovirus), we have found little evidence for the existence of hitherto unknown conserved miRNAs shared by other herpesviruses, although many genomes are predicted to harbor large numbers of miRNAs at similar genomic locations.

While EBV and rLCV thus share even more miRNAs than previously thought, our results underline the notion that, in contrast to their cellular counterparts, viral miRNAs show an almost total lack of sequence conservation.

*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG) and Hamburger Stiftung zur Förderung der Krebsbekämpfung

Monitoring the Epigenetic Fate of Gammaherpesvirus Genomes

T. Günther (1), U. Tessmer (1), A. Grundhoff (1) (1) Heinrich-Pette-Institute, Hamburg, GermanyLatent Infection with Kaposi´s Sarcoma associated Herpesvirus (KSHV) is etio-logically linked to the development of several tumors, including Kaposi`s Sarcoma (KS) and primary effusion lymphoma (PEL). Latency is a phase of the viral life-cycle during which viral gene expression is restricted to a small number of genes, and although epigenetic modifications are very likely to be of fundamental importance for latency establishment, their formation in the context of a de novo infection has hitherto not been analyzed.

We have developed a system to globally monitor the epigenetic status of KSHV genomes using a custom-designed, high resolution microarray. We have also devel-

Experimental Outline and Analysis of MeDIP Experiments. (A) Flow chart depicting the outline of MeDIP expe-riments. Genomic DNA is isolated from KSHV-infected or mock-infected cells. DNA from mock-infected cells is subsequently mixed with either unmethylated KSHV-bacmid DNA (neg. control) or bacmid DNA which has been fully methylated in vitro (pos. control). The samples are sheared, subjected to methylated DNA immun-precipitation, differentially labeled with Cy5 and Cy3, and analyzed on two color custom microarrays which carry the KSHV genome in high resolution. Data analysis, normalization and visualization is performed with custom developed software. A screenshot of the software interface is shown in (B).

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opped software for the refinement and normalization of the microarray data, as well as to provide graphic visualization of modified viral genomes. These tools were employed to analyze the epigenetic status of KSHV genomes at various timepoints following a de novo infection of endothe-lial SLK cells, using immunoprecipation of DNA via 5´-methylcytidine-specific anti-bodies (MeDIP) or antibodies specific for modified histones (ChIP protocol) to provide input for array analysis.

We find that de novo infecting episomes indeed adopt a characteristic and highly distinct DNA methylation profile with astonishing similarity to viral genomes in latently tumor-derived PEL cells. This is also true for deposition of acetylated histones, which showed a genome-wide pattern nearly complementary to DNA methylation. Furthermore, DNA methyla-tion appears to evolve over much longer time periods than histone modifications, suggesting the latter serve as an initial signal which subsequently triggers CpG methylation patterns to reinforce the latency program. Our approach, for the first time, allowed the global analysis of the 'epigenome' of de novo infecting KSHV genomes in a temporal and spatial manner. The data from our analysis will shed light on the molecular mechanisms underlying latency establishment as well as reactivation and lytic cycle induction.

*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG)

A requirement for Drosha in the Maintenance of Kaposi's sarcoma-associated Herpesvirus Latency

T. Christalla (1), U. Tessmer (1), A. Grundhoff (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

Kaposi’s Sarcoma associated Herpesvirus (KSHV) is the etiological agent of several tumors, including Kaposi`s Sarcoma (KS) and primary effusion lymphoma (PEL). The cells in PEL tumors as well as in PEL-derived cell lines are latently infected with KSHV. Latency is a phase of the viral lifecycle during which viral gene expression is restricted to a small number of genes. In contrast, during the lytic cycle the full com-plement of KSHV genes is expressed, culminating in the production and release of viral progeny and death of the host cell. The molecular mechanisms of latency estab-lishment are unclear, but it has been hypothesized that viral miRNAs might contrib-ute to the maintenance of latency via the silencing of lytic genes. We therefore have

The epigenetic status of KSHV episomes in PEL cell lines and de novo infected SLK cells. (A) Schematic Representation of the KSHV genome. Open reading frames in forward or reverse orientation are depicted as yellow or blue block arrows, respectively. (B) CpG frequency across the KSHV genome. (C) Global DNA methylation patterns of KSHV genomes in de novo infected SLK cells after establish-ment of stable latency. Note that the patterns are not a pure function of CpG content of individual loci. (D) DNA methylation patterns of KSHV genomes isolated from the tumor-derived PEL cell line BCBL1. (E) Histone acetylation patterns across the KSHV genome in BCBL1 cells. The patterns form a near perfect complement of the observed DNA methylation patterns.

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analyzed the effects of inhibiting the expression of Drosha (a RNAse III like enzyme which initiates maturation of miRNAs by excising pre-miRNA hairpins from their precursor transcripts) in the PEL-derived cell line BCBL-1. Indeed, shRNA-mediated knockdown of Drosha resulted in rapid and efficient induction of the lytic cycle, with an efficiency that was comparable to treatment with chemical agents such as phorbol esters or histone deacetylase inhibitors (which are commonly used to induce the lytic cycle in PEL cells). Surprisingly, however, knockdown of Dicer did not result in a similar induction of the lytic cycle. Like Drosha, Dicer is an RNAse III like enzyme, which processes pre-miRNA hairpins and is required for the production of mature miRNAs. During miRNA processsing, Dicer acts downstream of Drosha, and our data therefore indicate that the role of Drosha in the maintenance of KSHV latency is distinct from its function in the miRNA pathway. Recent data suggest that such a role may be the direct negative regulation of host transcripts which harbor pre-miRNA like hairpins. These transcripts are cleaved and directly destabilized by Drosha (hence a mode of regulation independent of Dicer), but the excised pre-miRNA like structures do not feed into the miRNA pathway. We are currently work-ing to establish the identity of cellular transcripts which are destabilized by Drosha and, upon stabilization by Drosha knockdown, induce lytic KSHV replication.

*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG)

Drosha knockdown induces lytic KSHV replication. (A) phase contrast (PC, top panel) and immunofluorescence staining for the viral lytic cycle specific DNA polymerase processivity factor (PF) encoded by ORF59 (lower panel) in the PEL-derived cell line BCBL1 after shRNA-mediated knockdown of Dicer or Drosha. (B) Higher magnification of Drosha knockdown cells with additional staining for the latency associated nuclear antigen (LANA) which is expressed during latent as well as lytic infection. (C) and (D) quantitative RT-PCR of transcripts encoding PF (C) or the immediate early protein ORF50 (D) at various timepoints post transduction with lenti-viruses encoding shRNAs that target Drosha or Dicer, or with a control lentivirus (pSico), or in cells which had been mock infected (control).

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A FACS-based FRET assay to analyze interactions between viral and cellular proteins in living cells

C. Banning (1), A. Düsedau (1), M. Warmer (1), R. Reimer (1), H. Hohenberg (1), F. Kirchhoff (2), M. Schindler (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) University of Ulm, Institute of Virology, Ulm, Germany

Most methods to study protein interactions in biological systems require protein purification or lysis and destruction of the cellular milieu. One of the few non-invasive techniques for protein interaction studies in living cells is Försters reso-nance energy transfer (FRET). In general, FRET measurements are done via fluo-rescence microscopy, which has serious limitations. For example it is difficult to quantify and compare results of multiple measurements or to ana-lyze large amounts of samples and cells. Here, we present a robust flow cytometry based FRET assay that allows to quantify protein interactions in a large number of cells. We applied this system to analyze the dynamic interactions of the human and simian immun-odeficiency virus (HIV and SIV) Nef proteins as well as the HIV-1 Vpu protein with various cellular host factors. Amongst others, our data suggest that - in strict contrast to HIV-1 Nefs (Na7, NL4-3) – SIVmac Nef strongly interacts with CD3. Furthermore we demon-strate direct binding of HIV-1 Vpu (NL43) to CD4 and tetherin (CD317), which has recently been shown to inhibit the release of progeny virions from infected cells. Currently we further develop our assay to screen for protein-protein interactions in high-throughput to generally identify unknown pro-tein interactions in living cells.

HIV-1 and SIV Nef both colocalize with CD3 in 293T cells transfected with pECFP-CD3 and pEYFP-Nef, respectively (upper panel). In contrast to this, only SIV-Nef interacts with CD3 as revealed by quantitative FACS-based FRET analysis (lower panel).

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Inhibition of Transferrin-Receptor Uptake by SIV and HIV Nef-alleles

M. Widder (1), S. Gundlach (1), F.X. Gobert (2), P. Benaroch (2), F. Kirchhoff (2), M. Schindler (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institut-Curie, Paris, France (3) University of Ulm, Institute of Virology, Ulm, Germany

The HIV-1 Nef protein contributes to the damaging high levels of immune acti-vation associated with T-cell loss and AIDS progression and is therefore com-monly considered a virulence factor. However, we recently found that Nef-alleles derived from asymptomatic naturally simian immunodeficiency virus (SIV) infected monkeys and the less pathogenic HIV-2 block the responsiveness of virally infected T-cells to activation and may protect the infected host against

damaging high levels of immune activation and loss of CD4+ T cells.. Further analysis revealed that Transferrin receptor (TfR) levels were elevated in virally infected primary T-cells express-ing Nef-alleles from SIV and HIV-2 but not from HIV-1. This effect was verified in 293T, THP-I and Jurkat T cells, as well as in infected primary lymphocytes and macrophages. Nef-expression alone was sufficient to induce upregulation of TfR. Nef-mediated inhibition of T-cell activation did not correlate with TfR upmodula-tion. Mutational analyses con-firmed that both represent inde-pendent Nef functions and SIV Nef upregulates TfR by a N-terminal AP2-binding motif. Internalization experiments

revealed that high TfR levels are a result of reduced receptor endocytosis follow-ing Nef expression. Possible consequences in vivo could be lower levels of intrac-ellular iron and reduced virus production. Our results show that upregulation of TfR is an independent Nef function that may contribute to the asymptomatic or attenuated course of infection observed in naturally SIV-infected monkeys and HIV-2 in humans.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Model of differential manipulation of iron-uptake by HIV and SIV nef-alleles. SIV and HIV-2 Nef inhibit TfR-internalization and might therefore decrease cellular iron-levels. In strict contrast, HIV-1 Nef has been reported to downmodulate HfE what should lead to an increase in TfR-internalization and hence iron-uptake. AP-2: adaptor-protein 2; CCV: clathrin-coated-vesicle.

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Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I Correlates with Loss of CD4+ T Cells in Natural SIV Infection

M. Schindler (1,6), J. Schmökel (1), A. Specht (1), H. Li (2), J. Münch (1), M. Khalid (1), D. Sodora (3), B. Hahn (2), G. Silvestri (4,5), F. Kirchhoff (1)(1) University of Ulm, Institute of Virology, Ulm, Germany (2) University of Alabama at Birmingham, Birmingham, Alabama, USA (3) University of Texas Southwestern Medical Center, Dallas, Texas, USA (4) Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia, USA (5) University of Pennsylvania, Philadelphia, Pennsylvania, USA (6) Heinrich-Pette-Institute, Hamburg, Germany

Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protec-tive effect remain unclear. To further assess the role of Nef in nonpatho-genic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 ani-mals with low (<500) CD4+ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effec-tor and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts ren-dered T cells hyper-responsive to activation, similar to those of HIV-1. Our data sug-gest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Correlation between Nef-mediated CD3 and MHC-I downmodulati-on in virally infected PBMCs. Correlation between the percentages of CD4+ T-cells in SIVsmm-infected SMs and n-fold downmodulati-on of CD3 and MHC-I. Assays were performed in PBMCs and stati-stical analysis was performed using the PRISM 4.0 package.

Virus-Host Interaction

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Single Nef Proteins from HIV-1 Subtypes C and F Fail to Up-Regulate Invariant Chain Cell Surface Expression but are Active for other Functions.

G. Turk (1), S. Gundlach (2), M. Carobene (1), M. Schindler (2), H. Salomon (1), P. Benaroch (3)(1) National Reference Center for AIDS, Buenos Aires, Argentinia (2) Heinrich-Pette-Institute, Hamburg, Germany (3) Institut-Curie, Paris, France

The HIV-1 Nef protein plays a major role in viral immunopathogenesis, modulat-ing surface expression of several immune receptors, altering signal transduction pathways and enhancing viral infectivity, among other activities. Nef also exhibits great intersubtype diversity but most studies have only been focused on Nef pro-teins from the subtype B. Thus, little is known about the functional capacities of non-subtype B Nef proteins in host cells. We investigated cell surface regulation of MHC-I, MHC-II, the MHC-II-associated chaperone invariant chain (Ii), CD4, CD3 and CD28 in cells transfected or infected with five different Nef-alleles includ-ing one HIV-1 Subype C and F allele. No significant difference among the Nef proteins regarding CD3, CD28 and MHC-II downregulation was observed. The NefC showed a slightly, yet significant, diminished capacity to downregulate MHC-I in all cells, as well as to down-regulate CD4 in Jurkat cells and PBMCs. Strikingly, the two alleles from NefC and NefF were unable to up-regulate the Ii chain both in transfected and infected cells. Moreover, internalization rate of sur-face Ii chain was slightly affected by NefC and NefF, whereas it was drastically reduced by NefB. Nef domains known to be involved in Ii chain up-regulation were conserved among the five alleles analyzed here. In summary, we identified two pri-mary HIV-1 NefC and NefF-alleles that are selectively impaired for Ii-upregulation and which may help to elucidate the mechanism of this Nef function in the future. It will be important to determine whether the observed differences are HIV-1 sub-type dependent and influence viral immunopathogenesis.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

*Supported by Stiftung Neuroviraler Erkrankungen (NVE)

Human Immunodeficiency Virus Type 1 Nef Expression Prevents AP-2-Mediated Internalization of the Major Histocompatibility Complex Class II-Associated Invariant Chain

H. Toussaint (1), F-X. Gobert (1), M. Schindler (2), C. Banning (2), P. Kozik (3), M. Jouve (1), F. Kirchhoff (4), P. Benaroch (1)(1) Institut-Curie, Paris, France (2) Heinrich-Pette-Institute, Hamburg, Germany (3) University of Cambridge, Cambridge CB2 0XY, UK (4) University of Ulm, Institute of Virology, Ulm, Germany

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells.

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However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodi-fied by Nef expression, further indicating that Nef regulates Ii chain internaliza-tion via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infec-tion. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell

Distribution of internalized anti-Ii chain MAb and Tf in the presence of Nef. HeLa-CIITA cells transfected with the bicistronic plasmid Nef NL4-3 (expressing Nef and GFP) or Nef-STOP (negative control expressing GFP only) were allowed to bind at 0°C with either Tf-Alexa Fluor 647 or the Ii chain-specific MAb. Cells were then shifted to 37°C for 5 min and fixed, and the Ii chain-specific MAb was revealed with Cy3-labeled secondary antibodies. Confocal sections of the indicated stainings are presented together with their overlays.

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surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Siglec expression on chimpanzee T-cells might contribute to the asymptomatic course of lentiviral infection in human primates

H. Ströver (1) and M. Schindler (1)(1) Heinrich-Pette-Institute, Hamburg, Germany

Siglecs (sialic acid-binding immunoglobulin-like lectins) belong to the Sialic acid-recognizing IG-superfamily lectins. They consist of an amino-terminal V-set immunoglobulin domain, which binds to sialic acid, and a variable number of C2-set immunoglobulin domains. Most Siglecs have an ITIM (immunoglobulin receptor family tyrosine-based inhibitory motif) and/or ITIM-like motif with which they are proprosed to block cellular activation. Siglecs are prominently expressed on immune cells, mainly of the leukocyte and the macrophage lineage. However, there are great species-specific differences which correlate with suscep-tibility to AIDS.

HIV-1 was transmitted into the human population from Chimpanzees which carry a virus closely related to HIV-1 (called SIV, simian immunodeficiency virus). Interestingly, in Chimpanzees experimentally infected with HIV-1 the virus establishes infection and replicates, but there is no AIDS progression observed. Thus, Chimpanzee are most likely genetically resistant towards HIV-1 infection. In contrast to humans, Chimpanzees express high levels of Siglecs on their T-cells, the main target cells of HIV-1. Siglec expression on those cells could dampen the cellular activation which is essential for efficient HIV-1 rep-lication. Therefore, the Siglec receptor familiy could be an important host fac-tor preventing CD4+ T-cell depletion and AIDS progression in the non-human Chimpanzee host.

So far, we confirm that different members of the Siglec family are able to suppress cellular activation of human Jurkat T-cells and primary T-cells. Mutation of the ITIM resulted in increased cellular activation, which demonstrates that Siglecs constitutively inhibit T-cell signalling via this motif. Currently, we investigate the effects of Siglec-expression on productive HIV-1 infection and replication.

HIV-1 budding and persistence in macrophages

C. Banning (1), B. Holstermann (1), H. Hohenberg (1), M. Schindler (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

HIV-1 infected macrophages accumulate viral particles in large intracellular vacuoles that originate from the lysosomal pathway. Those particle accumulation are infectious over weeks and represent viral reservoirs which can not be elimi-nated by the immune system. Strikingly, those vesicles do not fuse with lysosomes

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because of lack of acidification. The mechanism underlying this defect in acidifi-cation are largely unknown. Additionally, it is matter of intense debate if those viral particle accumulations originate from direct budding into intracellular vesicles or are reinternalized virions which have previously assembled at the plasma membrane. To address this question, we generated R5-tropic HIV-1 con-structs carrying GFP in the GAG region (iGAG) and infected primary macro-phages. Live-cell imaging is performed to track GAG and assembly and budding of newly synthesized virions. Furthermore we generated iGAG viruses with single knock-out mutations in nef, vpu, vpr and env. The latter constructs are used to investigate if acidification of lysosomal virus containing vesicles is pre-vented by one of these viral protein and as a consequence allows HIV-1 to persist in macrophages.

*Supported by Stiftung Neuroviraler Erkrankungen (NVE)

Upper panel: ultrastructural analysis of HIV-1 infected macrophages, showing large vesicles inside the cell (left). Viral particles are present inside those vesicles (right). Lower panel: HIV-1 GAG localization in different cell types visualized by GFP expression. Macrophages, primary T-cells and 293Ts were infected with viruses expressing GFP in the GAG-gene. Representative confocal images are presented.

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A serine phosphorylation site in HIV-1 Vpu is required for efficient counteraction of tetherin and replication in macrophages but not in ex vivo human lymphoid tissue

M. Schindler (1,2), D. Rajan (2) C. Banning (1), A. Iwanski (1), P. Wimmer (1), A. Specht (2), D. Sauter (2), F. Kirchhoff (2)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) University of Ulm, Institute of Virology, Ulm, Germany

The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and enhances the release of progeny virions by counteracting a restriction factor named tetherin (CD317). The latter effect is cell type dependent most likely due to differential levels of endogenous tetherin. Here, we show that mutation of a serine phosphorylation site at position 52 (S52A) disrupts Vpu-mediated degradation of CD4 and impairs its ability to antagonize tetherin. Nevertheless, the S52A substitu-tion did not reduce the ability of Vpu to enhance HIV-1 replication or CD4+ T-cell depletion in human lymphoid tissue. In strict contrast, HIV-1 expressing the S52A Vpu replicated only poorly in macrophages. The reason for these diverse effects of the S52A mutation on HIV-1 replication might be that activated CD4+ T-cells, that are the major target cells in lymphoid tissues, express substantially lower levels of tetherin than macrophages. Thus, our data suggest that a reduced ability of Vpu to counteract tetherin has differential effects on HIV-1 replication in CD4+ T cells and macrophages, the two most important cell types for viral replication in vivo.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

*Supported by Stiftung Neuroviraler Erkrankungen (NVE)

HIV-1 Vpu S52A replicates efficiently in lymphoid tissue but not in macrophages. Left panel: cummulative p24 production over 15 days in tissues from eight donors infected with the indicated HIV-1 variants. Values are given as percentages compared to cultures infected with NL4-3 wild-type (100%). Shown are means +/- SEM. Right panel: cummulative reverse transcriptase (RT) production by macrophages infected with the NL4-3 variants over a 20 day period. Values give averages +/- SEM of macrophages from three different donors infected with two independent virus stocks. Abbreviations: N-, Nef-defective; U-, Vpu-defective; S52A, VpuS52A; PSL, photon-stimulated luminescence; HLT, human lymphoid tissue; MDM, monocyte-derived-macrophages.

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Viral determinants for AIDS-progression in pediatric HIV-1 subtype B and C infections

C. Banning (1), H. Ströver (1), N. Eickel (1), A. Iwanski (1), P. Walker (2), L. Morris (2), M. Sornsakrin (3), R. Ganschow (3), M. Schindler (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) National Institute for Communicable Diseases (NICD), Johannesburg, South Africa (3) University Medical Center Hamburg-Eppendorf, Ambulance Clinic of Immune defects, Hamburg, Germany

The course of pediatric AIDS progression is characterized by high viral titers accompanied by increased levels of immune activation and immune cell dysfunc-tion. However, the viral determinants which are responsible for these effects are largely unknown. Accumulating data from in vitro experiments demonstrated that the HIV-1 Nef and Tat proteins lead to hyperactivation of virally infected T-cells. Furthermore, Nef was reported to cause B-cell dysfunction and hence hypergammaimmunoglobulinemia. The goal of our project is to assess the rele-vance of this in vitro findings in two cohorts of pediatric HIV-1 subtype B and C infections. In close collaboration with the UKE and the NICD we assembled blood samples and viral isolates from >50 HIV-1 subtype B and C infected chil-dren. The course of disease progression and clinical parameters are available for the majority of the patients enrolled in our study. HIV-1 nef and tat genes from all patient viral isolates will be amplified and analyzed for their capability to hyperactivate t-cells and to induce cytokines to stimulate uninfected bystander cells. Additionally, we will analyze multiple parameters which have been previ-ously suggested to be important for disease progression. The data from the func-tional analysis of pediatric Nef and Tat proteins will be correlated to various clinical parameters from the patients, to elucidate viral determinants which are important for AIDS-progression.

Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1 Is Restricted by both PML and Sp100

R.D. Everett (2), C. Parada (2), P. Gripon (3), H. Sirma H (1), A. Orr (2) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) MRC Virology Unit, Glasgow, U.K. (3) INSERM U522, Rennes, France

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expres-sion and plaque formation in limited passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organiser of cellular nuclear sub-structures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild type levels. In this study we report that

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depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression, and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modifi-cation undoubtedly play major roles in the regulation of HSV-1 infection, we found that pharma-cological interference with his-tone deacetylase activity was una-ble to complement the defect of ICP0 null mutant HSV-1 in either normal or PML depleted human fibroblasts. These data lend fur-ther weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and Stiftung für neuro-virale Erkrankungen

Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

A. Funk (1), M. Mhamdi (1), H. Hohenberg (1), J. Heeren (2), R. Reimer (1), C. Lambert (3), R. Prange (3), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Hamburg, Germany (3) Johannes-Gutenberg-University, Mainz, Germany

The identity and functionality of biological membranes is determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of bio-logical membranes favouring the formation of detergent-resistant microdo-mains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-sol-uble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increas-es the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral

HSV DNA is very commonly associated with PML Nuclear Bodies.

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envelope protein L is not signifi-cantly impaired. Infectivity and density of viral particles are par-tially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes is not significantly impaired, in con-trast to their release from endo-somes. We therefore conclude that viral, but not host choles-terol is required for endosomal escape of DHBV.

*Supported by Deutsche For-schungsgemeinschaft (DFG)

Strong and selective inhibitors of hepatitis B virus replication among novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues

E. Matthes (2), A. Funk (1), I. Krahn (2), K. Gaertner (2), M. von Janta-Lipinski (2), L. Lin (1), H. Will (1), H. Sirma (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) Max-Delbrück-Center of Molecular Medicine, Berlin, Germany

Novel N(4)-hydroxy- and 5-methyl-modified beta-L-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. beta-L-2',3'-Didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (beta-L-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective con-centration [EC(50)], 0.03 microM), followed by beta-L-2',3'-dideoxy-3'-thia-N(4)-hydroxycytidine (EC(50), 0.51 microM), beta-L-2',3'-dideoxy-N(4)-hydroxycytidine (EC(50), 0.55 microM), and beta-L-5-methyl-2'-deoxycytidine (EC(50), 0.9 microM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the beta-L-cytidine derivatives was also assessed. In accordance with the cell culture data, beta-L-Hyd4C triphos-phate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 microM. The cytotoxicities of some of the 4-NHOH-modified beta-L-nucleo-sides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH(2) group. The 50% cytotoxic concentrations for beta-L-Hyd4C in HepG2 and HL-60 cells were 2,500 microM and 3,500 microM, respectively. In summary, our results demonstrate that at least beta-L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Cholesterol dependent entry steps of DHBV.

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Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

M. Mhamdi (1), A. Funk (1), H. Hohenberg (1), H. Will (1), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

Formation of enveloped viruses involves assembly and budding at cellular membranes. Here we elu-cidated the morphogenesis of hepadnaviruses on ultrastructur-al and biochemical level using duck hepatitis B virus (DHBV) as a model system. Formation of virus progeny initiates at the endoplasmic reticulum (ER), and is conserved in vitro and in vivo. The morphogenesis proceeds via membrane-surrounded vesicles containing both virions and sub-viral particles (virus particles-containing vesicles, VCVs). VCVs are generated and maintained by reorganization of endomem-branes accompanied by a striking disorganisation of the rough ER. VCVs are novel organelles with

unique identity and properties of ER, intermediate compartment, endosomes, and multivesicular bodies. VCVs are dynamic structures whose size and shape are regulated by both fusion and fission. In conclusion, our data indicate a reorganisation of endomembranes during DHBV infection resulting in the biogenesis of novel organelles serving as multifunctional platforms for assem-bly and budding of virus progeny.

*Supported by Studienstiftung des deutschen Volkes and Stiftung neurovirale Erkrankungen (NVE)

Model of DHBV morphogenesis.

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Avian Hepatitis B Viruses: Molecular and Cellular Biology, Phylogenesis and Host Tropism

A. Funk (1), M. Mhamdi (1), H. Will (1), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV) share several fundamental features: both viruses have a partially double-stranded DNA genome which is replicated via an RNA intermediate. The coding open reading frames (ORFs) overlap extensively. In addition, the genomic and structural organiza-tion as well as replication and biological characteristics are very similar in both viruses. It is worth to mention that most of the key features of hepadnaviral infection were first discovered in the DHBV model system and subsequently confirmed for HBV. However, there are also several differences between human HBV and DHBV.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Life cycle of hepatitis B viruses.

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Efficiency of DHBV infection is restricted by an abortive entry pathway leading to degradation of the majority of incoming viral particles in vitro

A. Funk (1), M. Mhamdi (1), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

After endocytic entry into host cells, viruses exploit various strategies to bypass degradative compartments within the host cell. Hepadnaviruses do so by escap-ing from the early endosome presumably with the help of a translocation motif in the surface protein. Surprisingly, this escape is quiet inefficient, most of viral particles are not able to translocate into the cytosol. Here, we determined the overall stability of viral particles during entry and show that the majority of incoming viral particles is degraded. Kinetics of degradation differs for viral surface protein L and DNA. L has a half life of ca 1 h after viral entry and deg-radation depends on activity of specific cellular proteases and proteasome. In contrast, viral DNA is 24 h after entry about ten times reduced. Disruption of microtubules preventing infection led to further increased instability of viral DNA after this time period. Statistically, about 1 virion per cell ‘survives!’. Three days after infection, this single viral genome is amplified about 10 times. Taking into account that only a minority of hepatocytes is infected, the incoming rcDNA molecule is productively at least 100-fold amplified. Infection experi-ments with a mix of genotypically different DHBV largely confirmed the data. Taken together, our data show that the majority of viral particles is degraded after entry into host cells before escape into the cytosol and infection establish-ment. Our study indicates that efficiency of DHBV infection is determined by an abortive entry pathway leading to degradation of the majority of incoming viral particles.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Secretion of duck hepatitis B virus occurs via exocytosis

M. Mhamdi (1), A. Funk (1), H. Hohenberg (1), R. Reimer (1), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

Duck hepatitis B virus (DHBV) assembles and buds into virus particles con-taining-vesicles (VCVs) generated by the reorganization of the rough endoplas-mic reticulum (rER) and harbouring marker proteins of the ER-Golgi interme-diate compartment (ERGIC), endosomes and multivesicular bodies (MVBs). In this study, we have investigated the intracellular pathway followed by DHBV to secrete progeny virus using three well known inhibitors of the secretory path-way: Brefeldin A (BFA), monensin, and 20°C block. We found that viral secre-tion was drastically inhibited by BFA which induced the accumulation of viral particles within large VCVs near the nucleus and in close proximity to the cell surface. The other two inhibitors only slightly inhibited virus release and dur-ing the 20°C no accumulation of virus particles within the TGN was observed. We conclude that DHBV was transported to the cell surface by a Golgi-

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independent pathway. Using electron microscopy in conjunction with live con-focal microscopy, we showed that VCVs serves as transport carriers that are directed to the cell surface were they liberate 40-80 virions and 46.000 SVPs per hepatocyte and per hour via exocytosis.Taken together these data together with our previously published data indicate that VCVs are multifunctional platforms for assembly, budding, intracellular transport, and secretion of newly formed virus particles.

*Supported by Deutsche Forschungsgemeinschaft (DFG), Studienstiftung des deutschen Volkes und Stiftung für neurovirale Erkrankungen

Model of DHBV secretion.

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Naturally occurring adefovir resistance mutation rtI233V selected under adefovir therapy

O. Schildgen (2), C. Olotu (1), A. Funk (1), B. Zöllner (3), M. Helm (4), H. Sirma (1)(1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Bonn, Institute of Medical Microbiology, Immunology and Parasitology, Bonn, Germany (3) University Medical Center Hamburg-Eppendorf, Institute of Med. Microbiology, Hamburg, Germany (4) Praxis Abelein/Helm, Nürnberg, Germany

Recently, we reported on three clinical cases of chronic hepatitis B virus (HBV) infection resistant to adefovir (ADF) due to the pre-existing mutation rtI233V. Here, we further evaluated the clinical role of the rtI233V mutation. Serum samples from patients before and after ADF therapy were investigated for viral HBs antigen level and HBV DNA load. The RT region of the viral polymerase was analysed by sequencing of several cloned PCR products. We followed two of our previously reported cases of lamivudine and ADF resistant HBV infec-tion. Under tenofovir (TNF) treatment, one of the patients now has viral titers below the detection limit. The second patient developed renal failure under TNF therapy and was consequently treated with entecavir. Subsequently, the viremia increased from below the detection limit to 104 GE/ml. Interestingly, the rtI233V mutation partially converted to wildtype during entecavir therapy. In addition, two patients epidemiologically independent to the afore men-tioned cases were identified with ADF resistance associated to the rtI233V mutation that was selected during ADF therapy. The results strongly suggest that the mutation rtI233V is indeed responsible for ADF resistance. It can be selected during antiviral therapy but occurs also as natural variant prior to therapy. Inversely, cessation of selection pressure by ADF resulted in conversion of the mutation to the wildtype sequence. A therapy switch from TDF to entecavir in patients pretreated with ADF and lamivudine may decrease efficiency of entecavir treatment as compared to therapy naïve patients.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Cellular Determinants of HBV Replication

H. Boukari (1), H. Sirma (1) (1) Heinrich-Pette-Institute, Hamburg, Germany

Hepatitis B Virus is one of the most important human pathogens. These small DNA virus have a distinct liver tropism and causes liver-cirrhoses and primary liver-carcinoma. The HepaRG cell line. Upon their differentiation, HepaRG cells become susceptible for HBV infection. We found, that the HBV replication depends on the state of differentiation, in a positive manner. Furthermore, inter-action with the PML nuclear bodies and its components,, which have an impor-tant role in intrinsic antiviral response, were in focus. We found, that both HBV

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replication and gene expression are increased in PML- and Sp100-depleted cells, which are constitutive members of the PML-NB. Our results show that HBV replication depends on the differentiation state of the host cell and that PML-NB components PML and Sp100 are important factors modulating HB viral replica-tion and gene expression.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

High resolution electron microscopical (EM)-analysis of the distribution of TNF receptor 1 and the inhibition its internalization using immunogold stereo surface replica techniques

H. Dobner (1), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1), C. Schneider (1), W. Schneider-Brachert (2), M. Warmer (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institute for Medical Microbiology and Hygiene, University Regensburg, Germany

The adenoviral protein E3-14.7K is an inhibitor of TNF-induced apoptosis, but the molecular and structural mechanism underlying this protective effect has not been explained so far. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor–associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling com-plex (DISC). In the first step of our EM-study we analyse the exact distribution of TNF receptors at the plasma membrane surface of adenovirus–infected human cells applying the heavy metal immu-nogold surface replica technique. In a second step we will investigate by 3D ultrathin section reconstruction techniques the more complex intracytoplas-matical mechanisms that allow the adenovirus 14.7K protein to inhibit ligand-induced TNFR1 internalization.

Adenovirus-infected HeLa cells in a high resolution FESEM (overview).

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Cryotomography Ultrastructural analysis of cytomegalovirus tegu-ment protein interactions

H. Hohenberg (1), B. Holstermann (1), R. Reimer (1), C. Schneider (1), M. Warmer (1), M. Winkler (2) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institute for Infection Medicine, Kiel, Germany

Cytomegalovirus tegument pro-teins are critical constituents of the virus particle that play a role in particle assembly, virus entry and exit, establishment of infec-tion and immunomodulation. Based on an established map of protein interactions between tegu-ment proteins, which provides a scaffold for further definition of virus structure assembly and func-tion, the main focus of the investi-gations is the interaction between ppUL82 (pp71) and ppUL35. Both proteins localize to PML onco-genic domains and cooperatively transactivate the immediate-early enhancer of HCMV. In addition, deletion of the UL35 gene affects virus assembly by modulating nucleocytoplasmic localization of

other tegument proteins. Thus, ppUL35 is part of a cell-biological switch critical for immediate-early regulation and virus assembly. The use of Carbocell micro-reactors for sample preparation and ultrastructural analysis assures the perpetu-ation of virus components in situ during the whole investigation process. Cryofixation by high pressure freezing and deep temperature substitution are used to preserve the tegument proteins in their native state for further investiga-tion by immunogold-labelling and high resolution transmission electron micro-scopy.

*Supported by German Academic Exchange Service (DAAD)

Tomographic reconstruction of the different intracellular maturation steps of cytomegalovirus after cryoprocessing.

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Dynamic processes in organic and biological samples investigated with pump-probe experiments. Insights into the process of virus self-assembly in cells using a combination of microdiffraction and imaging techniques

H. Hohenberg (1), A. Meents (2), B. Reime (2), R. Reimer (1), T. Pakendorf (2) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) DESY, Hamburg, Germany

The bio-imaging and diffraction beamline P11 at DESY enables to investigate the structure of crystalline and non-crystalline biological materials at two dif-ferent end stations using an x-ray energy range between 3keV and 30keV with x-ray foci ranging from 10 nm to 250 nm. The DESY bio-imaging source allows the application of fluorescence tomography, phase contrast imaging and coher-ent diffraction imaging. In a collaboration project between the HPI-imaging group and the DESY the ultra-structure of cryo-processed viral protein assem-blies are investigated with a combination of x-ray imaging and diffraction techniques and compared with high resolution cryo-electron tomography.

Nanoparticle-Loaded Magnetophoretic Vesicles

M. Krack (1), H. Hohenberg (2), A. Kornowski (3), P. Lindner (1), H. Weller (1) and S. Förster (1) (1) University Hamburg, Institute of Chemistry, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

For many biomedical applications, the controlled uptake and release of active ingredients is of particular importance. In case of block copolymer vesicles, external stimuli such as changes in pH, temperature, UV-light, and concentra-tion of oxidizing agents can be used to trigger the release of substances in spe-cific therapeutic windows. A further attractive external trigger for diagnostics and therapeutic treatments are external magnetic fields. Magnetic nanoparti-cles have been employed for magnetic resonance imaging (MRI) and for mag-neto-thermal cancer therapy. The incorporation of magnetic nanoparticles into

A: Experimental hutch layout for beamline P11: All components are mounted on a granite block on which an intermediate focus, acting as a secondary source, is located. B: TEM image of cryoprocessed HIV capsid protein aggregates in a transfected E.coli cell.

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vesicles would create a versatile diagnostic and therapeutic tool that would be biocompatible, could be magnetically moved or targeted to specific cells or organs, and could be used for controlled release stimulated by external mag-netic fields. Block copolymer vesicles are particularly suited for this purpose because of their increased stability and bilayer thickness compared to lipid vesicles.

Size and Surface Effects on the MRI Relaxivity of Manganese Ferrite Nanoparticle Contrast Agents

U. I. Tromsdorf (1), N. C. Bigall (1), Michael G. Kaul (2), O.T. Bruns (3), M. S. Nikolic (1), B. Mollwitz (3), R. A. Sperling (4), R. Reimer (5), H. Hohenberg (5), W. J. Parak (4), S. Förster (1), U. Beisiegel (3), G. Adam (2) and H. Weller (1) (1) Institute of Physical Chemistry, University of Hamburg (2) Department of Diagnostic and Interventional Radiology, University Medical Center Hamburg-Eppendorf (3) Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf (4) Ludwig-Maximilians-University, Center of NanoScience (5) Heinrich-Pette-Institute, Hamburg, Germany

Superparamagnetic MnFe2O4 nanocrystals of different sizes were synthesized in high-boiling ether solvent and transferred into water using three different approaches. First, we applied a ligand exchange in order to form a water soluble polymer shell. Second, the particles were embedded into an amphiphilic polymer shell. Third, the nanoparticles were embedded into large micelles formed by lip-ids. Although all approaches lead to effective negative contrast enhancement, we observed significant differences concerning the magnitude of this effect. The transverse relaxivity, in particular r2*, is greatly higher for the micellar system compared to the polymer-coated particles using same-sized nanoparticles. We also observed an increase in transverse relaxivities with increasing particle size for the polymer-coated nanocrystals. The results are qualitatively compared with theoretical models describing the dependence of relaxivity on the size of mag-netic spheres.

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Real-time magnetic resonance imaging and quantification of lipoprotein metabolism in vivo using nanocrystals

O.T. Bruns (1), H. Ittrich (2), K. Peldschus (2), M.G. Kaul (2), U.I. Tromsdorf (3), J. Lauterwasser (1), M.S. Nikolic (3), B. Mollwitz (1), M. Merkel (4), N.C. Bigall (5), S. Sapra (5), R. Reimer (6), H. Hohenberg (6), H. Weller (3), A. Eychmüller (5), G. Adam (2), U. Beisiegel (1) and J. Heeren (1)(1) IBM II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (2) Department of Diagnostic and Interventional Radiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (3) Institute of Physical Chemistry, University of Hamburg (4) Asklepios Clinic St. Georg, 1. Department of Internal Medicine, Hamburg, Germany (5) Institute of Physical Chemistry and Electrochemistry, TU Dresden, Dresden, Germany (6) Heinrich-Pette-Institute, Hamburg, Germany

Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals have physical properties that are well suited for biomedical imaging. Previously, we have shown that iron oxide nanocrystals embedded within the lipid core of micelles show optimized characteristics for quantitative imaging. Here, we embed quantum dots and superparamagnetic iron oxide nanocrystals in the core of lipoproteins - micelles that transport lipids and other hydrophobic sub-stances in the blood - and show that it is possible to image and quantify the kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic magnetic resonance imaging. The lipoproteins were taken up by liver cells in wild-type mice and displayed defective clearance in knock-out mice lacking a lipoprotein receptor or its ligand, indicating that the nanocrystals did not influ-ence the specificity of the metabolic process. Using this strategy it is possible to study the clearance of lipoproteins in metabolic disorders and to improve the contrast in clinical imaging.

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IntroductionThe multitude and complexity of the networks operating within and between cells renders a cell vulnerable to dysregulation, either precipitated by exogenous stimuli or endogenous to the system. Even minor cellular disruptions can threat-en the fragile functional balance of a cell, leading to cellular damage at the level of individual organelles and/or at the level of the genome. To cope with such endogenous or exogenous stress situations, cells have developed a variety of pro-tective pathways that lead to either repair of the damage incurred or elimination of the damaged cell. Nevertheless, despite the elaborate protective arsenal of a cell, situations do occur that result in either transient or permanent alterations in cellular signaling characteristic of pathologic alterations. Important examples are viral infections and tumor development. In both conditions, cellular safeguards have failed, leading to the reprogramming of cellular pathways for the needs of viral replication or for successful tumor evolution. To understand these and other pathologic conditions, it is necessary to study in detail the basic mechanisms of cellular signaling, as well as the safeguard pathways insuring their proper function.

It is becoming increasingly evident that many pathological conditions lead to alterations in common cellular pathways or violate similar protective actions of the cell. In analyzing these pathways at the cellular level, the distinction between pathways dysregulated by viral infections or tumor evolution become irrelevant. Indeed, approaching the problems of cellular dysregulation from different angles clearly has synergistic effects. Thus the broad spectrum of projects addressing this topic serves the major goal of understanding the regulatory mechanism that govern the appropriate functioning of a cell, with the hope that understanding such mechanisms, and how they are deregulated under various pathologic con-ditions, will help us to identify handles for either preventing dysregulation or for curing the effects of cellular dysregulation at the level of the organism.

The Cellular Dysregulation Program has three core themes that are connected by this common research: 1) Cellular defense mechanism and tumor suppression; 2) Cellular controls of self-renewal, proliferation, and differentiation; and 3) Molecular mechanisms for “fine tuning” normal cell processes. All themes are also connected by the fact that both viral infections and tumor evolution impinge on one or, more often, many of these cellular control elements. This fact is also reflected in the different experimental approaches used within our Program to dissect the underlying mechanisms behind normal and abnormal cellular func-tions. Although traditionally the emphasis has and continues to be the prominent use of various viruses (e.g. adenovirus, polyoma virus, SV40, hepatitis virus and retrovirus) to identify and characterize cellular control mechanisms, there is also a strong deployment of animal models to mimic tumor growth in vivo, in which either viral elements or mutated cellular genes identified in patient samples are ectopically expressed. Mouse models hold the promise of yielding results that can be applied to tumor diagnosis and treatment and are highly adaptable to genetic

Programmbereich „Zelluläre Dysregulation“Research Program “Cellular Dysregulation“Head: Dr. Carol Stocking

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analysis for identifying causal relationships (within the normal environment). However, the success of many of the studies carried out within the Cellular Dysregulation program rely on new technical developments that extend the strength of cell biological experimentation in vitro. Here, the outstanding imaging resources available at our institute (fluorescent confocal microscopy, transmission electron microcscopy tomography, and correlative light / electron microscopy methods), as well several flow cytometry instruments (including a fluorescence-activated cell sorter) must also be mentioned.

In this biannual report, we have documented in form of brief abstracts many of the ongoing research projects, as well as successfully completed projects, in 2007 and 2008 within the program area “Cellular Dysregulation.” These abstracts have been grouped together on basis of the three core themes, which are briefly out-lined below.

A. Cellular defense mechanism and tumor suppression

The mammalian cell has evolved many mechanisms of protection against exter-nal assault (e.g. radiation, chemicals, infections, nutrient deficiencies, etc.) or inherent or sponataneous mutations in the cellular machinery. These mecha-nisms are part of a genetic program that is normally latent but is activated by poorly understood stress sensors. Activation of the defense program generally leads to cell-cycle arrest and DNA repair, however, in severely damaged cells the program triggers cell death through apoptosis and/or autophagy or in the execu-tion of a program that leads to a permanent growth arrest known as senescence. These responses are considered crucial tumor suppressive mechanisms, and thus are the targets of transforming viruses or spontaneous genetic mutations leading to cancer. Study of these defense mechanisms remains a major focus of the Cellular Dysregulation Program. These studies can be divided into three sections:

A1. Cellular target genes of viral transformation

Historically, viruses have been essential for the identification of cellular proteins and circuitries that are targeted during cellular transformation. Thus it was the analysis of DNA viruses that first led to the identification of p53, a central player in coordinating and executing cellular defense and protection mechanisms against endogenous and exogenous cellular stress situations. These types of studies are still very relevant, as demonstrated by ongoing work to dissect the transformation potential and cellular counterparts of gene products encoded by the E1B and E4 transcription units of the human adenoviruses or by the newly discovered Merkel cell polyomavirus (MCPyV), which is associated with a highly aggressive skin carcinoma.

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A2. Apoptosis, Senescence, and Autophagy

When damage accumulates irreversibly, mitotic cells rely on either of two mecha-nisms to avoid replication: permanent cell cycle arrest (cellular senescence) or cell death programs. Although apoptosis is the best-described form of programmed cell death, autophagy, which is a lysosomal degradation pathway essential for homeostasis, also contributes to cell death. Studies within our program address the important players in both apoptosis and senescence, as well as to identify cel-lular genes that counteract the activation of these programs in leukemia or other tumors, and thus important targets for therapy. Collaborative studies within our program also address the mechanisms in which bacteria subvert activation of the autophagy pathway as part of their virulence strategy.

A3. DNA repair mechanisms

Mechanisms by which defense strategies promote DNA repair remain an impor-tant focus of our work. A coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ATM and ATR protein kinases act as important regulators of the DNA-damage response by signaling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Our recent studies have provided new insights into the mechanisms by which the ATR-activated pathway delays cell cycle progression and irreversible inhibition of DNA replication involving the alternatively spliced p53 protein, termed ∆p53. Recent work has also implicated the SPOC1 protein in the ATM-dependent DNA-repair process. In addition to cellular protein, viral proteins (e.g. adenovirus E1B-AP5) also regulate ATR-induced DNA damage signaling pathways during infection, thereby promoting viral replication.

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B. Cellular controls of self-renewal, proliferation, and differentiation

There is increasing evidence that tumors are maintained by a subset of cancers cells that have the ability to self-renew and differentiate into the morphologically heterogeneous populations that are often observed in tumors, a concept first established for human myeloid leukemia. Thus understanding the complex cir-cuitry that regulates self-renewal, proliferation, and differentiation in tissue stem cells is important for deciphering the molecular events that occur in the genera-tion of the cancer stem cell. Our recent findings are presented in two subsec-tions:

B1. Gene transcription and chromatin modulation

Characterization of the genetic lesions in acute leukemia samples have demon-strated the importance of deregulated transcription factors in initiating neoplas-tic transformation by modulating the balance between self-renewal and differen-tiation. More recently, the growing importance of epigenetic changes in DNA and chromatin structure, which allows the accessibility of transcriptional factors at specific DNA-binding sites, has also been recognized. Several studies within the Cellular Dysregulation Program address the impact of hematopoietic trans-cription factors on transformation and differentiation. In addition, more recent studies assess the impact of mutant p53 proteins on transcription regulation to understand their gain-of-function role in tumorigenesis. Similarly, several find-ings support the role of SPOC1, a protein associated with aggressive ovarian cancer, as a pivotal regulator of chromatin structure during mitosis.

B2. Regulators of stem cells, proliferation, and differentiation

Another main focus of the research in this Program is the characterization of cancer stem cells and identifying the genetic lesions that are necessary to maintain them. In these studies both in vivo models of leukemia and mammary carcinoma are being used, as well as an in vitro model of embryonic carcinoma. These important studies have revealed a role for p53 and the MADS transcription factor Mef2c in regulating decisions of cell fate, which may have critical consequences for tumor/leukemia induction. Studies are also underway to assess the importance of TERT (encoding telomerase), which is normally down-regulated during differentiation and is postulated to be in important in maintaining the stem cell self-renewal potential. In addition to transcription factors, the role of activated receptor tyrosine kinase and their downstream signaling pathways are being investigated for their role in disrupting signals regulating proliferation and differentiation in leukemia.

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C. Molecular mechanisms for “fine tuning” normal cell processes

Proteins are the workhorse of several cellular processes and it is becoming increas-ingly clear from studies of viral and cellular proteins that their functional activation is regulated at several post-transcriptional levels that modulate their amount, localiza-tion, activity, and stability. These controls can occur both at the RNA and protein levels, and are the subject of several research endeavors within this program.

C1. Post-transcriptional and translational control mechanisms

Several cellular mechanisms by which protein amounts are controlled at the RNA level have been revealed by analysis of viral control elements and/or mechanisms of disease. This includes the CRM1-mediated nucleoytoplasmic transport of RNA, first identified by analysis of HIV-1, and the RNA chaperone La, an essential trans-acting factor in IRES-dependent translation initiation of picornaviruses. Our studies show that these mechanisms to control translation and elongation efficiencies are not restricted to viral RNA but are also used by a subset of cellular mRNAs. Similarly, novel insight into mechanisms that control mRNA maturation and stability has been gained by analysis of the consequence of point mutations or chromosomal translocations associated with disease. This includes nonsense mediated decay (NMD), a cellular mechanism of mRNA surveillance to detect nonsense mutations and prevent the expression of truncated or erroneous proteins, and R-loop formation during transcription, which can disrupt normal splicing and increase genomic instability, and eventually malignant cell transformation.

C2. Functional regulation by protein modifications

Post-translational modifications are key modulators of protein function. Several studies within our research program address the consequence of phosphoryla-tion, ubiquitination, and SUMOylation on the activity of viral and cellular pro-teins, and aim to identify the modifier proteins that regulate these processes. Importantly, the ability of p53 to bind to enzymes that mediate either phospho-rylation or ubiquitination is an important mechanism by which the specific activity of p53 as a transcription factor is controlled. Similarly, the adenovirus E1B-55K protein also exploits both ubiquitin and ubiquitin-like systems to target host cell proteins in transformation (e.g. SUMOylation of p53).

The following short reports document the high standard of research in this pro-gram area and the significant progress made during the last two years in the individual projects. The large number of publication in scientific journals with a high impact factor best supports this statement.

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Research ProjectsA. Cellular defense mechanismsA1. Cellular target genes of viral transformationCell transformation by human adenoviruses

B. Härtl (1), T. Sieber (1), P. Blanchette (3), P. Wimmer (1), E. Kremmer (4), T. Dobner (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (3) McGill University, Department of Biochemistry, Montreal, Canada (4) Institute for Molecular Immunology, Helmholzzentrum München, Germany

Human adenoviruses (Ads) are classified as DNA tumor viruses by virtue of their ability to cause tumors when inoculated into rodents. Two classes of oncogenic human Ads can be distinguished based on the types of tumor they elicit in ani-mals and the viral oncoproteins responsible for their tumorigenic potential. All members of the oncogenic subgroups A and B cause mostly undifferentiated sarcomas at the site of injection, while two members of the otherwise non-onco-genic subgroup D (Ad9 and Ad10) elicit exclusively estrogen-dependent mam-mary tumors following subcutaneous or intraperitoneal injection in female rats. Following the initial discovery of their oncogenic potential in animals it soon became apparent that human Ads provide an excellent experimental tool to ana-lyze fundamental events of normal and malignant cell growth. In particular, their ability to oncogenically transform primary mammalian cells in culture, which are then capable of initiating tumor growth in a susceptible host animal, has been of immense value in elucidating events in cellular and viral growth control. Much of our current understanding of the molecular mechanisms underlying virus-medi-ated oncogenic transformation has derived from the study of human Ads and the viral gene products involved in transformation. These studies led to the identifi-cation of the adenovirus oncoproteins, which in turn have served as molecular probes to resolve many fundamental aspects of cellular gene expression, cell cycle control and programmed cell death.

Today, it is well established that cell transformation by subgroup A and C Ads is a multistep process involving the cooperation of sev-eral gene products encoded in transcription units E1A, E1B and E4, as well as epigenetic changes resulting from viral DNA integra-tion. While E1A is clearly the key player in subgroup A and C Ad-mediated cell transformation and oncogenesis, it is becoming increasingly clear that other gene

The genomic organization of Ad5. The linear double-stranded genome is depicted in the center as a thin line, with the inverted terminal repeats (ITR) at each end: lengths are marked in map units. Transcription units are shown relative to their position and orien-tation in the Ad5 genome. Early genes (E1A, E1B, E2A, E2B, E3 and E4) are indicated by black bars. Genes expressed at intermediate times of infection (IX and IVa2) are indicated by gray bars. Open rectangles denote introns. Late genes (L1 - L5) produced from the major late promoter (MLP) all contain the tripartite leader (TPL) at their 5’ ends and are shown at the top of the figure. Small arrows denote virus-associated RNAs I and II (VA RNAs).

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products substantially affect the tumorigenic phenotype of the transformed cell. In particular, work in our group on the oncogenic potential of E1B and E4 gene products have now revealed new pathways that point to novel, general mecha-nisms of virus-mediated oncogenesis.

Products of early region 1B (E1B) from human Ads are capable of cooperating with Ad E1A to transform primary mammalian cells in culture. It is generally considered that the large E1B-55K products contribute to complete cell transfor-mation by antagonizing apoptosis and growth arrest, which primarily result from the induction and metabolic stabilization of the tumor suppressor protein p53 by Ad E1A. These growth-promoting activities correlate with their ability to act as a direct transcriptional repressor that is targeted to p53-responsive promoters by

Protein interaction domains and functional motifs in the Ad5 E1B-55K protein. Domains involved in nucleo-cytoplasmic shuttling, RNA-binding, transcriptional repression and nuclear localization (NLS) are indicated on top. Conserved amino acid residues (cysteine histidine, serine and threonine) are indicated below. Mutations that affect various processes shown on the right are indicated by boxes and triangles, and numbers refer to their positions in the E1B amino acid sequence. Arrows indicate the high conservation of the USP7- and CRM1-binding motif and the SUMO-conjugation site, as illustrated by the absolute complexity diagram (bottom).

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binding to p53. In addition it has been hypothesized that the mode of action of Ad E1B-55K products during transformation may involve additional functions and other protein interactions.

Over the past years we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transfor-mation are encoded within several patches of the 55K primary sequence including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino acid substitutions (C454S/C456S) created a 55K mutant protein, which was substantially reduced in transforming activity. Interestingly, the same mutations affected neither binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transfor-mation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA double-strand break repair complex and/or its ability to assemble multiprotein aggre-gates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.

Further support for this model originates from genetic and biochemical analyses to assess role of E1B-55K related proteins E1B-156R, E1B-93R and E1B-84R in the transformation process. Results from these assays show that E1B-156R, which com-prises the 79 amino-terminal and 77 carboxy-terminal amino acids of E1B-55K, also enhances focal transformation of primary rat cells in cooperation with E1A. Since E1B-156R seemed unable to relocalize p53 and inhibit its transactivating function, it must be assumed that it contributes to transformation independently of repression of p53-stimulated transcription. Furthermore, we found that E1B-156R contains a functional transcriptional repression domain and binds the cellular apoptosis regulator Daxx. Taken together, these analyses introduce E1B-156R as a viral oncoprotein that contributes to cell transformation by a novel mechanism, which likely involves binding to and modulation of the cellular Daxx protein.

*Supported by Deutsche Forschungsgemeinschaft and in part by Wilhelm Sander-Stiftung

RNA interference as a tool to study adenovirus E1A/ E1B-mediated cell transformation

E. Koyuncu and T. Dobner Heinrich-Pette-Institute, Hamburg, Germany

In this study we evaluated the use of RNA interference (RNAi) as an experimental tool to assess the role of known and potential E1B-55K interaction partners on E1A- plus E1B-mediated transformation of primary baby rat kidney (BRK) cells. Results from these studies demonstrate that coexpression of E1A with p53-specific short hairpin RNAs (shRNAs) efficiently induced the formation of transformed clones which could be established into permanent cell lines. Surprisingly, shRNA-

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p53 appeared to be more efficient to promote E1A-induced focus formation than E1B-55K. More importantly, the total number of transformed cells was further greatly increased when both plasmids were simultaneously cotransfected with E1A, suggesting that E1B-55K and shRNA-p53 can synergistically promote focus-formation in the presence of E1A. These data demonstrate that silencing of cellular targets of E1B-55K through RNAi can be used to evaluate their role in cell trans-formation and provide further support for the idea that the viral oncoprotein contributes to oncogenesis through p53-dependent and -independent pathways.

*Supported by Erich und Gertrud Roggenbuck-Stiftung

Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines

N. Fischer (1), J. Brandner (2), F. Fuchs (2) , I. Moll (2), A. Grundhoff (3) (1) University Medical Center Hamburg-Eppendorf, Institute of Med. Microbiology, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Institute of Dermatology, Hamburg, Germany (3) Heinrich-Pette-Institute, Hamburg, Germany

MCPyV in Merkel cell carcinoma (MCC) cell lines. (A) MCPyV copy numbers in MCC cell lines relative to GAPDH copy numbers, as determined by quantitative PCR. All values are depicted as mean ± standard deviation of three independent experiments. (B) Detection of MCPyV genomes in three MCC lines by southern blotting. (C) Schematic representation of the MCPyV LT-Ag and the position of point mutations (oval shapes) or deletions (grey boxes) which leads to premature termination of translation.

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Merkel cell carcinoma (MCC) is a highly aggressive tumor of the skin which predominantly afflicts elderly and immunocompromised patients. The recently discovered Merkel cell polyomavirus (MCPyV) is believed to play a causative role in the pathogenesis of MCC. This hypothesis is supported by several lines of evidence: (i) MCPyV is detected with a frequency of ~80 % in MCC tissues, (ii) the tumors harbor clonally integrated MCPyV genomes, indicating that integration preceeds tumor formation and (iii) the gene encoding large T-antigen (LT-Ag) gene of integrated MCPyV genomes unequivocally harbor mutations which selectively abrogate its ability to support viral replication while preserving its ability to target the cellular tumor supressor Rb.

While tumor derived cell lines represent valuable tools for the in vitro study of MC carcinomas in general and the possible contribution of MCPyV in particu-lar, only a few MCC cell lines have hitherto been established and only one of these has been reported to be infected with MCPyV. We have analyzed 3 existing and 4 newly established MCC cell lines for the presence, integration pattern and copy number of MCPyV. In five cell lines, MCPyV specific sequences were detected. In three of these lines, Southern Blotting and/or quantitative PCR sug-gest clonal integration of concatemeres of MCPyV genome. All three lines con-tained the LT-Ag truncating mutations characteristic for MCPyV genomes found in primary MCC tumors. The premature stop codons were localized in the region between the regions coding for the Rb binding and origin binding domains of LT-Ag.

In contrast, the remaining two lines had average copy numbers equal to or less than 1/50 per cell, indicating that the cultures consist of a mixture of infected and uninfected cells. This may reflect outgrowth of original MCPyV-positive

Classification of Merkel cell carcinoma (MCC) cell lines. (A-B) Immunofluorescence staining of CgA in the variant MCC cell line MCCL-6 (A, A´) and the classic line MCCL-3 (B, B´). (A, B) epifluroescence picture of CgA staining. (A´, B´) overlay of epifluorescence pictures of CgA (red) and DAPI (blue) staining. (C-F) Morphological subtypes of MCC cell lines: (C) MCCL-5 representing type I, (D) MCCL-7 representing type II, (E) MCCL-11 representing type III and (F) MCCL-12 representing type IV. Note that all cell lines are grown on feeder layer which is also seen in (C-F). Bars: (A-C): 20 µm, (C-F): 100 µm.

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tumor cells along with uninfected bystander cells during cell line establishment, or elimination of viral genomes upon cultivation in vitro. The latter would argue for the presence of a selectional pressure against MCPyV in some, but not all lines, a hypothesis which is currently under investigation.

MCC cell lines are conventionally categorized as “classic” or “variant” and further divided into four subtypes, based on expression of neuroendocrine markers such as chromogranin A (CgA) and cell as well as colony morphology. While it has been suggested that the presence of MCPyV might promote a classic phenotype, we identified MCPyV-positive as well as -negative lines of the classic variety, indi-cating that the distinguishing features are either inherently independent of viral infection or have become so in the course of tumorigenesis and/or cell line estab-lishment. We are currently investigating the contribution of MCPyV infection to the maintenance of the transformed status of the various MCC cell lines, as well as the transforming potential of the shortened large T-Ag proteins in heterolo-gous systems.

*Supported by Bundesministerium für Gesundheit (BMG)

A2. Apoptosis, Senescence, and Autophagyp53-Rb2/p130 crosstalk in cellular senescence

W. Bohn (1), H. Helmbold (1), N. Kömm (1), U. Galderisi (2) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) University of Naples, Italy

Cellular senescence is a basic response of diploid mammalian cells to genotoxic stress. It is activated to withdraw DNA damaged cells irreversibly from the cell cycle in order to prevent uncontrolled proliferative activity. The response is induced by intrinsic stress signals, arising from shortening of telomeres or from the activity of viral and cellular oncogenes. The response can also be induced artificially by extrinsic signals, such as irradiation or the application of chemotherapeutic drugs. The appearance of senescent cells specifically in premalignant tumor lesions suggests that cellular senescence acts to suppress the transition from the benign to the malignant stage in tumor development.

We aim at identifying signaling pathways that determine induction and mainte-nance of the senescent growth arrest, and are of prognostic relevance in tumor treatment. It is known that p53 and retinoblastoma (Rb) proteins play the most important role in the senescence response. Functional inactivation of these proteins or their signaling pathways abrogates the senescent growth arrest. Using rat glioma cells as an initial experimental cell system we identified Rb2/p130 as the retinoblas-toma protein specifically interacting with p53 in this response. The cells are pre-cisely arrested between the cyclin-E to cyclin- A mediated steps in the cell cycle. While remaining arrested, the cells gradually adopt a senescence phenotype, which is indicative of reaching an irreversible stage of growth arrest. Irreversibility is based on epigenetic changes leading to irreversible chromatin silencing. We extended the studies to human tumor cells subjected to DNA damaging treatment. We obtained evidence that the Rb2/p130 - p53 crosstalk is of general relevance for activation of the senescence response. siRNA mediated knock-down of Rb2/p130 but not of Rb1

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impaired arrest activation and led to progression of cells into S-phase of the cell cycle. The same outcome was obtained when p53 or its downstream target CDK-inhibitor p21WAF1 were inactivated. Thus human tumor cells can activate cellular senescence in response to DNA damaging treatment even when they lack a func-tional Rb1 signaling pathway, provided they have a wild-type p53 gene status.

Activation of senescence pathways by unfavorable culture conditions could also be responsible for the limited life span of mesenchymal stem cells (MSC) in vitro, a major obstacle for their use in basic molecular and cell therapy studies. Using rat mesenchymal cells as a model system, we have determined the relationship between adopting a senescent phenotype and changes in stemness markers and DNA repair mechanisms. We found a strong and general reduction in the expression of all genes involved in DNA repair, and a loss of stemness markers. This suggests that DNA dam-age sustains the senescence process in MSCs in an auto-loop-like pathway. Entry into senescence occurred with characteristic changes in Rb expression patterns. Rb1 and p107 gene expression decreased during in vitro cultivation. By contrast, pRb2/p130 became the prominent Rb protein. This suggests that Rb2/p130 could be a marker of senescence or that it even plays a role in triggering the process in MSCs.

*Supported by Deutsche Krebshilfe

Dissecting the mechanism of apoptosis-resistance in hematopoietic malignancies expressing wild-type p53

A. Engelmann (1), K. Schulz (1), D. Speidel (1), G. Bornkamm (2) W. Deppert (1), C. Stocking (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) GSF, Munich, Germany

Tumors that have acquired resistance against death stimuli constitute a severe problem in the context of cancer therapy. To determine genetic alterations that favor the development of stress resistant tumors in vivo, we took advantage of polyclonal tumors created by retroviral infection of newborn Eλ-myc mice, in which the retroviral integration acts as a gene mutagen to enhance tumor pro-gression. Tumor cells were cultivated ex vivo and exposed to gamma-irradiation prior to their re-transplantation into syngenic recipients, thereby providing a strong selective pressure for pro-survival mutants. Secondary tumors develop-ing from treatment resistant cell clones were analyzed for retroviral integration sites to reveal potential candidate genes whose dysregulation confer the selec-tion advantage. In addition to the anti-apoptotic gene Bcl-xL, we identified the Gadd45β locus to be a novel common integration site in these stress-resistant tumors. Enhanced expression of Gadd45β was observed in secondary tumors, but not in cells from primary non-stressed parental tumors. In accord with a thus far undocumented supportive role of Gadd45β in tumorigenesis, we showed that Gadd45β over-expressing NIH3T3 cells form tumors in NOD/SCID mice. Interestingly, and in contrast to “classical” anti-apoptotic factors, high Gadd45β levels did not protect against c-myc, UV or gamma-irradiation induced apoptosis, but conferred a strong and specific survival advantage against serum withdrawal.

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To identify other candidate genes that protected tumors from stress-induced apoptosis, several tumor pairs (radiation sensitive vs. resistant tumors derived from the same parental tumor) were screened by profiling expression arrays. Three candidate genes were identified, whose expression was upregulated in radiation resistant tumors by fivefold. Analysis demonstrated that these were all normally upregulated after apoptosis stimulation. Complementary DNA were isolated and expressed in hematopoietic cells and their ectopic expression is cur-rently being tested for their ability to protect from radiation or other stress response.

*Supported by Deutsche Krebshilfe

FLASH meets nuclear bodies: CD95 receptor signals via a nuclear pathway

E. Krieghoff (1), K. Milovic-Holm, (2) T.G. Hofmann (1) (1) German Cancer Research Center, Heidelberg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

The CD95 receptor signals via assembly of a multi-protein complex termed death-inducing signaling complex (DISC) which triggers activation of receptor-bound caspase-8/FLICE molecules. Most cells (type II cells) depend on a mito-chondrial amplification pathway to commit apoptosis upon CD95 activation. The caspase-8-binding protein FLICE-associated huge protein (FLASH) has been previously implicated in the regulation of caspase-8 activation at the DISC.

Model of the apoptotic and non-apoptotic functions of FLASH. (A) Nuclear body associated FLASH is shuttled into the cytoplasm upon activation of CD95, dependent on caspase activity and CRM-1. Cytoplasmic FLASH then associates with mitochondrial caspase-8 thereby promoting apoptosis. This function can be inhibited by adenoviral protein E1B19K that associates with both caspase 8 and FLASH at mitochondria. (B) Non-apoptotic functions of FLASH. After TNFalpha treatment FLASH may translocate into the nucleus and interacts there with hormone receptors and receptor coactivators to modulate transcription. FLASH is important for Cajal body architecture, interacts with NPAT and enhances histone gene transcription and S-phase progression; right: FLASH promotes TNFalpha-mediated activation of NFkappaB signalling by forming a complex with TRAF2, leading to degradation of IkapppaB, nuclear accumulation of NFkappaB and expression of its target genes.

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However, recent findings demonstrated that FLASH is a Cajal body component and regulates progression through S-phase of the cell cycle in the nucleus. Our recent work identified FLASH as binding partner of the PML nuclear body (PML NB) constituent Sp100 and demonstrated that FLASH partially localizes to PML NBs. Upon CD95 activation FLASH exits the nucleus and translocates to mitochondria where it meets caspase-8 to promote its activation. Our find-ings reconcile conflicting views on FLASH localization and its role in apoptosis regulation, and suggest that CD95 signals via a nuclear pathway. These findings have several implications for understanding FLASH function.

* Supported by Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe

Functional p53 is required for effective telomerase inhibition in BCR-ABL-positive CML cells in vitro

U. Brassat (1), S. Balabanov (1), H. Sirma (2), C. Günes (2), M. Braig (1), D. Bali (3), K. Borgmann (3), T.H. Brümmendorf (1) (1) University Medical Center Hamburg-Eppendorf, Hubertus Wald University Cancer Research Center, Hamburg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany (3) University Medical Center Hamburg-Eppendorf, Radiobiology and exp. Radiooncology, Hamburg, Germany

Telomeres consist of repeat structures such as (TTAGGG)n in vertebrates and are localized at the end of chromosomes. Replication-dependent telomere shortening due to the end-replication problem can be counteracted by upregulation of an endogenous reverse-transcriptase called telomerase. Increasing evidence suggests that critical telomere shortening results in genetic instability which may promote tumour evolution and telomerase activation during which critically short telom-eres are stabilised and ongoing tumour growth is facilitated. In Chronic myeloid leukemia (CML) the high turnover of the malignant clone is driven by the onco-gene BCR-ABL and leads to accelerated telomere shortening in chronic phase (CP) compared to telomere length in healthy individuals. Telomere shortening has been demonstrated to be correlated with disease stage, duration, prognosis and response to molecular targeted treatment. Despite of the accelerated telomere shortening observed, telomerase activity is increased in CP CML and further upregulated with progression of the disease to accelerated phase or blast crisis (AP/BC). To investi-gate the effect of telomerase inhibition on BCR-ABL-positive cells, we expressed a dominant-negative mutant of hTERT (vector pOS DNhTERT-IRES-GFP) in K562 cells. The cells were single sorted and clones in addition to bulk cultures were long term expanded in vitro. The expression of the transgene DNhTERT was monitored by the expression of GFP and function of DNhTERT was analyzed by measure-ment of telomere length (by flow-FISH) and telomerase activity (TRAP assay). Evaluation of these parameters showed the following patterns of growth kinetics and telomere biology in individual clones: Two clones lost telomere repeats and were transiently delayed in growth kinetics but eventually escaped from crisis without loss of GFP expression (indicated by a re-increase in telomere length and growth rate, group A) Three other clones lost GFP expression after initial and sig-

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nificant telomere reduction indicating loss of the transgene (group B). Finally, telomere length and growt0h kinetics of two remaining clones and of the bulk culture cells remained unaffected by expression of DN-hTERT (group C). Of note, none of the clones analyzed either died or entered cell cycle arrest. Further analyses of one clone of group A revealed impaired DNA damage response indicated by two fold increase in number of γH2AX foci in comparison to control cells. Moreover, the expression pattern of genes involved in DNA repair was significantly altered (Dual chip®). Network analysis of the altered genes using MetaCore® software confirmed p53 as a key regulator in signaling of DNA damage in these cells. CML blast crisis cell lines such as K562 are typically negative for functional p53 and p16INK4A. Induction of p53 in cells with critically short telomeres (telomere length 4-5 kb) leads to immediate induction of apoptosis while vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is dependent on the presence of functional p53 in BCR-ABL-positive cells, which argues in favor of applying these strategies preferentially in CP as opposed to BC.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Invasin- and β1-Integrin-dependent uptake of bacteria is coupled to the activation of autophagy and suppressed by Yersinia enterocolitica

A. Deuretzbacher (1), N. Czymmeck (1), R. Reimer (3), H. Hohenberg (3), K. Trülzsch (2), J. Heesemann (2), M. Aepfelbacher (1), and K. Ruckdeschel (1) (1) Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, Hamburg, Germany (2) Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Munich (3) Heinrich-Pette-Institute, Hamburg, Germany

Autophagy is a central lysosomal degradation process that is essential for the maintenance of cellular homeostasis. Autophagy-related processes also pro-mote separation and degradation of intracellular microorganisms, which con-tributes to the development of innate and adaptive immunity. Here, we inves-tigated the impact of the enteropathogenic gram-negative bacterium Yersinia enterocolitica on autophagy in infected macrophages. Autophagy was analyzed by monitoring processing of the marker protein LC3 and by visualizing autophagosome formation. Our results show that Yersinia virulence factors differentially target the autophagic response in macrophages. Avirulent yersiniae that were defective in virulence plasmid-associated type III protein secretion efficiently activated autophagy. The autophagy-related events includ-ed the formation of autophagosomes, processing of LC3, redistribution of GFP-LC3 to bacteria-containg vacuoles, and the segregation of intracellular bacteria by autophagosomal compartments. Autophagy followed engulfment of the bacteria by macrophages and specifically involved the Yersinia outer membrane protein invasin. The autophagic events depended on engagement of β1-integrin receptors. This delineates a novel pathway through which the sensing of a bacterial microbe triggers autophagy. Wild type Y. enterocolitica on the

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contrary inhibited bacterial uptake and the activation of autophagy in a type III protein secretion dependent manner. The autophagy responses elicited by rapamycin and chloroquine were also suppressed by wild type Yersinia infection. This indicates global targeting of the autophagic system by Yersinia-dependent immunomodulation which involves several Yersinia outer proteins (Yops) mobilized by type III secretion. We propose that the subversion of autophagy is part of the Y. enterocolitica virulence strategy that supports bacterial survival when β1-integrin-dependent internali-zation into macrophages is dele-terious for the pathogen and conducive for host immunity.

A3. Mechanisms DNA repairRegulation and function of the stress-induced polymerase α-primase-wtp53 complex in the ATR-mediated intra-S-phase checkpoint

G. Rohaly, A. Mena-Nunez, S. Dehde, H. Will, I. Dornreiter Heinrich-Pette-Institute, Hamburg, Germany

In UV-damaged S-phase cells, it is inopportune to initiate DNA synthesis when template strands are damaged because mutations and chromosomal aberrations are induced through the process of replication of damaged DNA. One important response to replication stress is the activation of the ATR-mediated intra-S check-point, which signals through the combined action of the Chk1-Cdc25A-cyclin E-Cdk2 and the ∆p53-p21-cyclin A-Cdk2 pathways. The damage pathways delay further progression through the cell cycle, inhibit origin firing, and promote lesion repair. Although the transcriptional activity of p53 is repressed in attenu-ated S-phase cells, complete inhibition of DNA replication is only observed in wtp53, but not in mutp53 primate cells. Our investigations revealed that wtp53 interacts exclusively with origin-competent DNA polymerase α-primase (Pol α) only during the damage induced intra-S checkpoint. The p53 interacting domain is located in the N-terminus of polymerase α, containing the Cdk phosphoryla-tion sites that are involved in initiation control. A prerequisite for Pol α-wtp53 complex formation is ATR-∆p53-p21-mediated down regulation of cyclin A-Cdk2 activity, while high cyclin A-Cdk2/1 activity leads to complex dissociation. Moreover, in vitro data demonstrate that wtp53 inhibits the primase but not the

Internalized Yersiniae in a murine J774A.1 macrophage cell in TEM.

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polymerase α activity of the replicase. Thus, data suggest that the intra-S check-point induced interaction of wtp53 with Pol α prevents origin firing to allow unperturbed repair of genotoxic lesions before the onset of DNA replication.

*Supported by Deutsche Forschungsgemeinschaft

Repair fidelity of UV-induced lesions depends on ATR and the p53 status

V. Deing, G. Rohaly, A. Mena-Nunez, S. Dehde, H. Hildebrandt, H. Will, I. Dornreiter Heinrich-Pette-Institute, Hamburg, Germany

In UV-damaged cells, the photoproducts [6-4]PP are regarded as the principal UV-induced lesion that jeopardize replication fidelity and thus should activate the intra-S checkpoint response. The fact explains why the [6-4]PP lesions are much more efficiently repaired than CPDs and >80 % of the [6-4]PPs are removed from human fibroblast DNA within three hours after UV irradiation. Therefore, with such rapid repair of damage, addition of an extra hour or two of repair before replication should have a significant impact on outcome. In a recent study of our own, we found that only in wtp53 cells onset of DNA repli-cation is delayed for six hours after treating the cells at the G1/S transition with a low dose of UV. Our data demonstrate that during the six-hour replication arrest, high repair activity is observed. Thus, it seems reasonable to assume that inhibition of origin firing is a mechanism needed to allow complete repair of [6-4]PP lesions and accordingly to prevent error prone bypass replication. Accordingly, we assumed that the ATR-∆p53-p21-cyclin A-Cdk2-mediated attenuation of S-phase progression and -mediated inhibition of origin-depend-ent initiation serves the purpose to enable repair of the genotoxic photoprod-ucts [6-4]PP prior to the onset of replication. The assumption was investigated by measuring the repair efficiency in UV-irradiated G1/S wtp53 CV1 and TO-CV-1-mut∆p53 cells, where Dox addition leads to inactivation of endog-enous ∆p53. Data demonstrated that in UV-irradiated G1/S CV-1 cells [6-4]PPs were completely removed after 6 h. In contrast, in damaged TO-CV-1-mut∆p53 cells, where attenuation of S-phase progression and extended inhibition of rep-lication is abrogated, the [6-4]PP lesions were removed in less than 2 h. Although the [6-4]PP lesions were removed just in time before replication was initiated, irradiated TO-CV-1-mut∆p53 cells responded with checkpoint activation in G2-phase. During attenuation of G2-phase progression, high repair activity was observed indicating that the accelerated [6-4]PP-repair process was executed at the expense of repair fidelity, leading to secondary DNA damage. The same results were obtained when ∆p53-transactivated p21 was knocked down by Dox-induced shRNAp21 expression. It is noteworthy that the CPD repair kinet-ic was not altered in UV-irradiated G1/S cells devoid of functional ∆p53 or p21. However, inactivation of endogenous ATR by dominant-negative effects severe-ly impaired repair of the photoproducts CPD and [6-4]PP in UV-irradiated G1/S TO-CV-1-ATRkd cells. Data strongly indicate that besides the ATR-activated ∆p53-p21-cyclin A-Cdk2 pathway, which delays S-phase progression and initia-

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tion of replication in favor of high fidelity [6-4]PP lesion repair, ATR is involved in enabling nucleotide excision repair in damaged S-phase cells in general by an yet unknown mechanism.

*Supported by Deutsche Forschungsgemeinschaft

In UV-damaged S-phase cells, delayed origin loading of Pol α depends on the ATR-∆p53-p21-cyclin A-Cdk2 pathway

G. Rohaly, V. Deing, H. Will, I. Dornreiter Heinrich-Pette-Institute, Hamburg, Germany

Our investigations demonstrated that the UV-induced ATR/Chk1 signaling pathway causes transient delays in S-phase progression and reversible inhibition of DNA replica-tion for 6 h. The key players of this pathway, which allow extended S-phase attenuation and inhibition of replication, are transcriptional active ∆p53 and transcriptional impaired wtp53. The transcriptional activity of ∆p53 leads to p21-mediated down regu-lation of cyclin A-Cdk2 activity, an activity that is required for S-phase progression and DNA synthesis. Down regulation of cyclin A-Cdk2 activity is a prerequisite for facilitat-ing interaction of the tumor suppressor wtp53 with hypophosphorylated, origin-com-petent Pol α, an event that correlates with inhibition of origin firing. However, it is not known if p53 binds to origin-bound Pol α or sequestrates Pol α in order to prevent origin loading. The question was addressed by using the ChIP-PCR method to deter-mine the time point of origin-associated hypophosphorylated Pol α in sham- and UV-treated CV-1 cells at specific time points post treatment. In order to establish ChIP-PCR for origin-associated proteins, the well-characterized component of the origin-recognition complex Orc2 was immunoprecipitated with an anti-Orc2 antibody from CV-1 cell lysates. Association of Orc2 with ors12 origin and non-origin was analyzed by PCR amplification. Results show that immunoprecipitated Orc2 contained only origin, but not non-origin DNA, indicating that the ors12 locus is suitable for analyzing origin interacting factors by ChIP-PCR in monkey CV-1 cells. Accordingly, investigations were performed to evaluate whether hypophosphorylated Pol α interacts with the ors12 origin during the UV-induced six-hour S-phase attenuation. For the immunoprecipitation, two monoclonal anti-Pol α antibodies were used: HP180-12, which recognizes hypo-phosphorylated, origin-competent Pol α and SJK132-20, which is specific for the cyclin A-Cdk-modified, origin-incompetent replicase. Results demonstrated that in mock treated CV-1 cells HP180-12-reactive Pol α was associated with the ors12 origin only in early S- (3 h post G1/S) but not in late S-phase (7 h post G1/S), confirming that all origins had fired at the end of the replicative S-phase. In contrast, in UV-irradiated CV-1 cells origin DNA was only recovered from HP180-12 immunoprecipitates at 7 h but not 3 h post irradiation, indicating that in attenuated S-phase cells origin loading of Pol α is prevented. In contrast, ChIP-PCR performed with sham-or UV-treated mut∆p53 expressing TO-CV-1 cells demonstrated that origin loading of HP180-12-reactive Pol α was not delayed, thus resembling the pattern that was derived from non-irradiated cells. In addition, data showed that in mock as well as in UV-irradiated CV-1 or TO-CV-1-Flag-mut∆p53 cells SJK132-20-reactive Pol α was never associated with the ors12 origin. The fact that origin loading of hypophosphorylated Pol α is prevented during the six-hour continuing UV-induced ATR-∆p53-p21-cyclin A-Cdk2-mediated S-phase attenu-

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ation suggests that p53 sequestrates and accordingly abrogates origin loading of the replicase. In addition, confocal microscopy was performed to examine chromatin asso-ciation of the HP180-12- and SJK132-20-reactive Pol α in mock- and UV-irradiated cells, which were pre-extracted at specific time points post treatment. Data demonstrated that loading of HP180-12-reactive Pol α onto chromatin is significantly delayed in UV-irradiated CV-1 but not in TO-CV-1-Flag-mut∆p53 cells and never co-localized with p53. However, it remains to be elucidated if in mutp53 expressing CV-1 cells origin loading of Pol α is also delayed during S-phase attenuation.

*Supported by Deutsche Forschungsgemeinschaft

Loading of HP180-12-reactive, origin-competent Pol α onto the ors12 origin in mock- or UV-irradiated CV-1 and TO-CV-1-Flag-mut∆p53 cells at specific times post irradiation.

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Dissection of the role of SPOC1 in DNA repair

A. Mund, G. Rohaly, I. Dornreiter, S. Schönfeldt, A. Mena-Nunez, S. Dehde, K. Reumann, H. Will Heinrich-Pette Institute, Hamburg, Germany

Chromatin modifications are important for all cellular processes that involve DNA, including transcription, DNA replication and repair to maintain genomic stability. One crucial event is the phosphorylation of histone H2AX (γH2AX) in damage-induced modification of chromatin structure, which links the recruitment of chromatin remodeling and repair complexes to sites of damaged DNA. Recently our investigations revealed that our newly discovered protein SPOC1 (Survival time associated PHD finger in Ovarian Cancer 1), a tightly cell cycle regulated chromatin associated protein, co-localizes with DNA repair proteins in response to DNA double strand breaks. Laser scanning microscopy demonstrated that SPOC1 co-localizes with γH2AX at nuclear foci shortly before the onset of DNA replication (G1/S-transiton). Additionally, we could show that the damage-induced SPOC1 foci formation depends on functional ATM. ATM is a member of the phosphoinositide 3-kinase-related kinases (PIKK), which activate a complex

Formation of ATM-dependent SPOC1 (green) and gamma-H2AX (red) foci in human primary fibroblasts after gamma irradiation. A/B: ATM mutants show no SPOC1 foci after 30min of 2Gy irradiation but still disperse g-H2AX foci formation. C/D: Wt cells show distinct SPOC1 foci that co-localize with gamma-H2AX foci.

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network involved in the DNA damage response pathways. Thus, our results strongly indicate that SPOC1 is a new component of the ATM-mediated DNA-damage response pathway that operates in the G1/S-checkpoint.

*Supported by Deutsche Krebshilfe and Müggenburg Stiftung

A role for E1B-AP5 in ATR signalling pathways during adenovirus infection

A. Blackford (1), R. Bruton (1), O. Dirlik (2), G. Stewart (1), A. Malcolm (1), R. Taylor (1), T. Dobner (2), R. Grand (1), A. Turnell (1) (1) CRC Institute for Cancer Studies, The University of Birmingham, UK (2) Heinrich-Pette-Institute, Hamburg, Germany

E1B-55K-Associated Protein 5 (E1B-AP5), is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here we reveal a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during adenovi-rus infection. Specifically, we show that E1B-AP5 is recruited to viral replication centers during infection where it co-localizes with ATR-Interacting Protein (ATRIP) and the ATR substrate, RPA32. Indeed, E1B-AP5 associates with ATRIP and RPA complex component, RPA70, in both uninfected and Ad-infected cells. Additionally, GST pull-downs show that E1B-AP5 associates with RPA compo-nents, RPA70 and RPA32, directly in vitro. Significantly, we have determined that E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting to note that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9 and Smc1 during infection, such that Ad12 pro-motes significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation, and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved to utilize different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implica-tions for regulation of ATR activity during the cellular DNA replication cycle or in response to DNA damage.

*Supported by Cancer Research UK

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B. Cellular controls of self-renewal, proliferation, and differentiationB1. Gene transcription and chromatin modulationModulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences

M. Brázdová (1), T. Quante (1), L. Tögel (1), K. Walter (1), C. Loscher (1), V. Tichý (2), L. Cincárová (2), W. Deppert (1), G.V. Tolstonog (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institute of Biophysics, Brno, Czech Republic

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of tran-scription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53R273H in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequenc-es are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53R273H targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

*Supported by the Deutsche Forschungsgemeinschaft (DFG), by EU FP6 and by the German-Israeli Foundation (G.I.F.)

Mutant p53R273H is targeted to the nucleus in U251 glioblastoma cells and associates with transcription factories.

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Mutant and wild-type p53 as modulator of global chromatin organization

T. Quante (1), A. März (1), L. Tögel, S. Hess (2), D.P.F. Möller (2), G. Warnecke (1), M. Kühl (1), C. Loscher (1), W. Deppert (1), G.V. Tolstonog (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Department of Informatics, University of Hamburg, Germany

Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to its wild-type counterpart, the mutant p53 (mutp53) protein is long-lived and has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the propensity for targeting to chromatin. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used tumor cell lines expressing endogenous and inducible mutp53 proteins as models to study the role of mutp53 in transcriptional regulation. Mutp53 has lost sequence specificity in DNA binding activity but retained the property to recognize DNA secondary structures, and based on our ChIP-Seq data is prone to interact preferentially with repetitive sequences possessing conformational flex-ibility. Therefore we assumed that mutp53 operates on the level of global chromatin organization rather than on modulating the expression of individual genes. In support, we found that differentially regulated genes frequently map to the same chromosomal locations or even are organized as physically-linked gene clusters. This implies that mutp53 might be a factor involved in regulating the recruitment of gene promoters to immobile transcription factories containing active RNA polymerases. To test this hypothesis, physical DNA contacts in several genomic loci encompassing co-regulated gene clusters were profiled using the chromosome conformation capture (3C) method. Preliminary results strongly support the proposed function of mutp53 in modulating the gene expression program at a higher level.

*Supported by Deutsche Forschungsgemeinschaft (DFG), by EU FP6, and by the German-Israeli Foundation (G.I.F.)

P53 as a potential epigenetic modulator of gene expression

J. Hildebrand (1), A. März (1), L. Tögel (1), T. Quante (1), S. Hess (2), D.P.F. Möller (2), G. Warnecke (1), M. Kühl (1), C. Loscher (1), W. Deppert (1), G. V. Tolstonog (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Department of Informatics, University of Hamburg, Germany

Transcription factors may act as dual-purpose regulators, controlling gene expression either via direct targeting of a gene or via (re)organizing higher-order chromatin struc-ture. In this regard, recent genome-wide studies of the p53 binding to DNA demon-strated that p53 binding not only is restricted to regulatory sequences around transcrip-tion start sites, but rather that binding events are widely distributed over the whole genome. Extending the term “epigenetics” to (i) transcription events outside of coding genes and (ii) regulatory reactions primarily based on nuclear (re)organization, we want to propose that the function of p53 in regulating the gene expression program includes also a role in epigenetic processes. In the frame of this hypothesis we tested the possibil-ity that p53 may regulate transcription of long interspersed nucleotide elements (LINEs),

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a family of transcriptionally potent repetitive sequences, via binding to their promoters. Around 12.000 full-length LINEs are dispersed throughout the human genome and can be potentially engaged by the transcription machinery. Most of the full-length LINEs are non-coding, whereas only 1 % has retained a coding potential for two proteins required for retrotransposition. LINE transcription is developmentally regulated, whereby the highest expression is observed during embryogenesis, in germ line cells and tumors. To test the putative transactivation function of p53 on the LINE transcription, we construct-ed an episomal eGFP-based reporter gene, which allowed us an accurate measurement of LINE promoter activity via eGFP fluorescence. In H1299 cells engineered for inducible expression of p53, we observed significant stimulation of the episomal LINE promoter, accompanied by a p53-dependent increase of endogenous LINE transcription. The latter observation was confirmed in 5-FU treated HCT116 (p53+/+) cells and the teratocarci-noma cell lines 2102Ep (human) and F9 (mouse), and could be reversed by p53-specific siRNA. Binding of p53 to endogenous LINE promoters was confirmed by ChIP and cor-related with the occupation of the respective promoters by RNA polymerase II.

Analyzing the transcriptome profiles of H1299 cells expressing Tet-inducible p53 we noted that many regulated genes frequently map to the same chromosomal locations or even are organized in physically-linked functional gene clusters. The number of genes in such co-regulated clusters correlated well with the time-dependent increase in p53 con-centration after beginning of Tet-induced ectopic p53 expression. To allow for a system-atic analysis of gene co-regulation, we programmed a script, which can match data from the most common commercial platforms to current genome annotation, search for the co-regulated genes based on variable criteria, and do cross-analysis between different samples to find groups of common co-regulated genes. By genome-wide ChIP-on-chip analysis we observed that significant number of co-regulated gene clusters was associated with the single p53 binding events, supporting our assumption that p53 is additionally involved in global epigenetic processes leading to modulation of the transcriptional activity of the whole chromatin domain.

*Supported by Deutsche Forschungsgemeinschaft (DFG), and EU FP6

Oncogenicity and expression profiling of SPOC1 in normal and tumor tissues

H. Staege (1), J. Weise (1), J. Hengstler (2), K. Milde-Langosch (3), C. Günes (1), S. Kinkley (1), U. Matschl (1), G. Mohrmann (1), H. Will (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) IfADo, Dortmund, Germany (3) University Medical Center Hamburg-Eppendorf, Institute of Gynpathology, Hamburg, Germany

We have analyzed the expression levels of the novel SPOC1 protein in different tissues of human and mouse origin. Highest expression of SPOC1 RNA and protein was detected in the testis and lower levels were found in all other tissues examined. In general, the level of SPOC1 expression correlated with the proliferative state of the tissue, indicating a pos-sible role for SPOC1 in normal cellular proliferation. Prompted by our initial observa-tion that high levels of SPOC1 RNA are correlated with poor survival and residual dis-ease in a subset of ovarian cancer patients, we extended our studies to investigate SPOC1

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protein expression in ovarian cancer. Consistent with the RNA studies, we found that SPOC1 protein levels were significantly increased in a subset of ovarian tumors. These findings sug-gest that SPOC1 may have oncogenic potential and could be a useful diag-nostic/prognostic marker for ovarian cancers. Studies to investigate SPOC1 expression in other types of cancer and to correlate its expression with other described tumour markers are ongoing.

Furthermore, preliminary evidence shows that cell lines with elevated SPOC1 protein expression gives rise to faster growing tumors than the parental cell lines alone in nude mice. A more extensive study evaluating SPOC1 overexpressing cell lines in

comparison to the parental cell lines with lower steady state levels of SPOC1 protein in nude mice are underway. These studies should provide conclusive evidence as to the oncogenicity of enhanced SPOC1 protein expression.

*Supported by Deutsche Krebshilfe and the Müggenburg Stiftung

SPOC1: A novel PHD containing protein modulating chromatin structure and mitotic chromosome condensation

S. Kinkley (1), H. Staege (1), G. Mohrmann (1), G. Rohaly (1), T. Schaub (1), E. Kremmer (2), A. Winterpacht (3), and H. Will (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) GSF-Research Center, München, Germany (3) University of Erlangen, Institute of Human Genetics, Erlangen, Germany

In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients which statis-tically correlated with poor prognosis and residual disease. These observations implied that SPOC1 may play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expres-sion as well as localization are regulated throughout the cell cycle. SPOC1 demonstrates a dynamic regulation during mitosis with increased expression levels and biphasic localiza-tion to mitotic chromosomes, arguing for a functional role of SPOC1 in mitotic proc-esses. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show via micrococcal nuclease (MNase) chro-matin digestion assays that SPOC1 expression levels proportionally influence the degree

Increased SPOC1 levels in a subset of ovarian carcinomas. SPOC1 protein levels were determined in lysates from normal ovary and from ovarian carcinoma tissues by immunoblotting. SPOC1 was detected with a rat monoclonal anti-SPOC1 antibody and β-actin served as loading control.

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of chromatin compaction. Collectively, our findings show that SPOC1 modulates chro-matin structure and that tight regulation of its expression levels and subcellular localiza-tion during mitosis are crucial for proper chromosome condensation and cell division.

*Supported by Deutsche Krebshilfe and Müggenburg Stiftung

Enhanced SPOC1 expression induces chromatin instability

S. Kinkley (1), H. Staege (1), K. Reumann (1), M. Cremer (2), A. Jauch (3), and H.Will (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) LMU Biozentrum, Department Biology II, Martinisried, Germany (3) Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany

SPOC1 is a novel chromatin associated protein that shows elevated expression levels and a dynamic regulation during mitosis. Recent results suggest that SPOC1 is important for proper chromatin architecture and condensation of mitotic chromo-somes arguing indirectly that it may also be important for genomic stability. Consistent with this possibility, several doxycyclin-inducible U2OS SPOC1 over-expressing cell lines became 100 % polyploid in the absence of doxycyclin after multiple passages in culture, due to a low level of leaky SPOC1 over-expression (2-fold) in these cell lines. The polyploidization of these cell lines were charted by FACS analysis and later confirmed by M-FISH. Furthermore, doxycyclin induction of SPOC1 over-expression (~30-fold) in these polyploid cell lines resulted in chromosome losses, inducing an aneuploidy-like state from a tetraploid intermedi-ate. These observations were reproducible also in Dld-1 cells, which had originally a

Overexpression of SPOC1 induces global chromosome condensation and this depends on its plant homeo domain (PHD).

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normal diploid chromosome count and a single stable chromosome translocation, demonstrating a global penetrance and potency of SPOC1. The underlying mechanism of SPOC1 induced polyploidization and chromatin instability is not yet clear, however it appears to involve the deregulation of key cyclins/cdk complexes and some of their downstream targets. Elucidating this mechanism is a main focus of our research program and is currently being actively pursued. Together these findings indicate that a tight regulation of SPOC1 expression levels is crucial for the maintenance of a normal diploid state in cells and therefore for genomic stability.

*Supported by Deutsche Krebshilfe and Müggenburg Stiftung

SPOC1: A novel chromatin remodeling protein?

S.Kinkley (1), H. Staege (1), N. Lohrengel (1), G. Mohrmann (1), K. Reumann (1), A. Eberharter (2), and H. Will (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) Adolf-Butenandt Institute of Molecular Biology, University of Munich, Munich, Germany

SPOC1 is a chromatin-associated protein with a described function in the regulation of mitotic chromatin architecture. A more detailed analysis of SPOC1’s relationship with chromatin revealed that it is capable of specifically interacting with DNA, histones and nucleosomes, arguing strongly for chromatin related function of SPOC1. To further clarify the specificity of SPOC1’s interaction with chromatin and to gain some insight into the potential role it plays there, we systematically evaluated its ability to bind dif-

Low level of SPOC1 overexpression results in 100 % polyploidization of cell lines. A. Immunoblotting of SPOC1 expression levels in wild-type U2OS cells and in two U2OS inducible SPOC1 clones (Cl. 5 and Cl.14) in their uninduced (UI) and induced (Ind.) states. Actin served as a loading control. B. FACS profiling of these cell lines shows that both U2OS SPOC clone 5 and clone 14 are 100 % polyploidy in comparison to the U2OS cell line. C. M-FISH analysis confirms that the U2OS SPOC1 clones are polyploidy in comparison to the parental U2OS and shows that chromosome losses occur when the SPOC1 clones are induced for SPOC1 over expression.

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ferentially modified histones. Recently there have been several publications describing PHD domains as the molecular reader of H3K4 di- and tri- methylation marks. Consistent with the literature, SPOC1 was found to specifically associate with di- and tri-methylated H3K4 in a PHD domain dependent manner. SPOC1 is also capable of binding to other modified histones however, in a PHD-domain independent manner, implicating that another as of yet undefined region in the SPOC1 protein is also capable of interacting with chromatin. Interestingly, transient over expression of the SPOC1 protein in multiple cell lines results in massive global hyper-condensation of chromatin in a PHD-domain dependent manner. This dramatic phenomenon results in the con-densation and global remodeling of the entire genome, similar to what is observed during mitosis. It is our current hypothesis, that SPOC1 may act either directly or indi-rectly to recruit condensing remodeling complexes to the chromatin and may therefore act as a molecular switch between a euchromatin state to a more heterochromatic state. Consistent with this possibility, SPOC1 is found to co-elute by column chromatography in three high molecular weight fractions of ~400 kDa, 800 kDa and 2000 kDa, arguing that SPOC1 is part of a greater protein complex. Elucidation of these complex members should help to clarify the biological functional roles of SPOC1 as well as potentially the mechanism of SPOC1 induced chromosome condensation. Therefore in this study we aim to understand the molecular context and consequences of SPOC1 on chromatin.

*Supported by Deutsche Krebshilfe and Müggenburg Stiftung

Mitotic Changes in SPOC 1 Localisation and Expression. SPOC1 changes its localization during the mitotic cell cycle as shown by immuno-fluorescence staining and by immuno-blotting using synchronized cells.

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The RUNX1 transcription factor: A gatekeeper in acute leukaemia

J. Cammenga (1,2), M. Fischer (1),), K. Weber (1,3), U. Bergholz (1), M. Ziegler (1), S. Roscher (1), U. Müller (1), C. Stocking (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, University of Lund, Sweden (3) University Medical Center Hamburg-Eppendorf, Center of BM-Transplantation, Hamburg, Germany

RUNX1 is one of the most frequently mutated genes in human acute leukemias and hematopoietic disorders. Located on chromosome 21, it is directly targeted by several chromosomal translocations in AML, with the t(8;21) being the most common, occurring at a frequency of approximately 15 %. Moreover, 20 % of pediatric B-cell acute lymphoblastic leukemia (ALL) carry the t(12;21), juxtaposing the RUNX1 gene with the gene encoding the ETS transcription factor ETV6. In addition to translocations, inactivating or dominant-negative mutations in the RUNX1 gene have been identified in 15 to 25 % of the relatively rare, minimally differentiated M0 AMLs, up to 25 % of myelodysplastic syndromes associated with AML

The RUNX1 gene is a frequent target of genetic disruption in acute leukemia. Specific chromosomal transloca-tions or point mutations involving the RUNX1 gene are found in acute myeloid or lymphoid leukemia with a distinctive phenotype. Depicted is the lineage and differentiation stage in which the bulk of the leukemia cells with a characteristic genetic mutation accumulate. The genetic mutations result in either fusion or mutated RUNX1 proteins, as depicted.

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development, and in pedigrees of familial platelet disorder with a propensity to develop AML. RUNX1 gene product (RUNX1 or often called AML1) is one of three members of the RUNX family of transcription factors that play pivotal roles in normal development. RUNX proteins bind DNA at sequence-specific motifs through a conserved Runt homology domain, which is also the interacting domain of a shared β-subunit that modulates nuclear localization and DNA binding. Significantly, disruption of the gene encoding the β-subunit is also observed in 5 % of AMLs.

Over the last few years, we have established mouse models for translocations t(8;21) and t(12;21) and, more recently, for mutated RUNX1 genes, using a retroviral-vector transduction protocol coupled with bone marrow transplantation. The coexpression of GFP in this model has allowed the evaluation of changes in the hematopoietic system before the onset of an overt leukemia, providing an excellent model to study the multiple steps of leukemogenesis. The evaluation of these mice have lead to the following conclusions: 1) intrinsic functions of the fusion protein or mutated protein play a determining role in the phenotype (myeloid vs lymphoid; early or late; T- or B-cell) of the ensuing leukemia; 2) whereas repression of direct target genes of RUNX1 is a common functional denominator of RUNX1 mutations, DNA-binding-independent functions of RUNX1 play an important role in leukemogenesis; 3) RUNX1 gene alterations alone are insufficient to induce a leukemia, but probably act as gatekeeper, permitting the outgrowth of a preleukemic clone, which is the target of secondary events.

More recent data have demonstrated that Runx1 forms large multiprotein complexes with several classes of chromatin modulators and plays a pivotal role in the initial chromatin remodeling during lineage speciation. We favor the hypothesis that Runx1 regulates B-cell specification by chromatin modulation of the PU.1 locus, in contrast myeloid differentiation per se does not require continued Runx1 expression, but Runx1 is required to modulate proliferation or anti-apoptotic stimuli. We are currently testing this hypothesis in Runx1 conditional mice. We are also extending our work to human hematopoietic cells, using a xenograft transplantation model (NOD/scid mice). Differences in the transcriptional control of the hematopoietic system between mouse and man are known, and may greatly influence the action of an altered transcription factor. For these studies we have developed lentiviral vectors that allow the efficient expression of a fluorescent marker gene (e.g. VENUS, tdTomato, EGFP, or Cyan), a potential oncogene, and a cassette to express short-hairpin RNA, suppressing gene expression Runx1 to determine its function in human hematopoiesis.

*Supported by Fritz-Thyssen-Stiftung, Deutsche José Carrera's Leukämie-Stiftung, and Deutsche Krebshilfe.

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B2. Regulators of stem cells, proliferation, and differentiationThe MADS transcription factor MEF2C is an important regulator of monopoiesis and the leukemic stem cell compartment in MLL-associated leukemia

A. Schüler (1), M. Täger (1), M. Forster (1), J. Löhler (1), M. Ziegler (1), S. Roscher (1), R. Delwel (2), E. Olson (3), R. Slany (4), C. Stocking (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) Dept of Hematology, Erasmus University, Rotterdam, The Netherlands (3) University of Texas Southwestern Medical Center, Dallas TX, USA (4) Friedrich-Alexander University Erlangen-Nürnberg, Germany

Multiple lines of evidence indicate that tumorigenesis is a multistep process originating from a single cell. In myeloid leukemia, the concept that a hematopoietic stem cell (HSC) is the first normal cell that becomes subverted in the leukemogenic process is gaining wide acceptance. Importantly, several recent in vivo studies have also demonstrated that a small number of the immediate progeny of this cell, which still share many of characteristics of the HSC compartment, are responsible for maintaining the leukemia and thus are denoted the leukemic stem cell (LSC) compartment. Understanding how a HSC is converted to a LSC is critical for establishing effective therapeutic intervention of acute myeloid leukemia (AML). We have used retroviral insertional mutagenesis to identify novel genes that regulate myeloid differentiation – and thus are potential targets of gene disruptions that establish the LSC. Two members of the MEF2 gene family were identified in this screen.

The MEF2 proteins are members of the MADS (MCM1-agamous-deficiens-serum response factor) family of transcription factors, which play key roles in pattern devel-opment in plants and coordinate diverse and important biological functions, including proliferation, differentiation, and apoptosis, in the animal. Importantly, the activity of

MADS-box proteins is profoundly influenced by developmental cues and signals from extracellular environment through several tran-scriptional and post-translational controls. The MEF2 subfamily, which consists of four members (A-D) and distinguishes itself with the presence of both a MADS domain and a conserved MEF2 domain, important for homo/and heterodimerization. In contrast to the accumulating evidence supporting the importance of MEF2 in neuronal and myogenic development and despite the fact that several MEF2 proteins are highly expressed in hematopoietic tissue, little is known about their role in myeloid and lymphoid development.

Mef2c is an important link between extracellular stimuli and intra-cellular controls. Our work has shown that cell fate decisions bet-ween the monocytic and granulocytic lineages are modulated by the Mef2c transcription factor upon activation through extracellu-lar signals (e.g. growth factors or bacterial infections). Activation of Mef2c upregulates expression of the bZIP transcription factor cJun. We postulate that cJun forms heterodimers with C/EBP proteins, thereby crippling their ability to regulate transcription of important granulocytic genes and thus favouring monocytic differentiation driven by the ets transcription factor PU.1.

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Using a retroviral gene transduction – bone marrow transplantation model we have been able to demonstrate the importance of MEF2C in AML development in susceptible mice. This analysis has also shown the importance of MEF2C in lineage commitment to the monocytic lineage, which has been corroborated by analysis of conditional Mef2c knock-out mice. cJun was determined to be an important down-stream target of MEF2C for this activity. Analysis of AML patient samples provided the insight that high MEF2C expression is found in myelomonocytic leukemia samples characterized by alterations of the bithorax orthologue MLL. Deletion of the Mef2c gene in MLL-transformed cells cripples their ability to induce leukemia in vivo and thus may play an important role in maintaining the LSC compartment.

*Supported by Deutsche José Carrera's Leukämie-Stfitung

Aldehyde dehydrogenase 1 (ALDH1) as a functional marker of cancer stem cells in a mammary carcinoma mouse model

F. Wegwitz (1), C. Maenz (1), J. Oehlmann (1), A. Diesterbeck (1), A. Preuß (1), G. Pilnitz-Stolze (1), A. Düsedau (1), A.-M. Kluth (1), U. Schumacher (2), G. Tolstonog (1), W. Deppert (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Dep. of Anatomy, Hamburg, Germany

Since the discovery that a small subset of self-renewing leukemia cells drive cancer growth and propagation about 10 years ago, the cancer stem cell theory has been extended to solid tumours, including breast cancer. However, despite the attractiveness of the cancer stem cell theory, most “cancer stem cells” are still only operationally defined and characterized by empirically established cell surface markers.

Several studies describe the expression of ALDH1, which can be easily detected by its fluorescent substrate Aldefluor®, as a prospective marker of mammary carcinoma stem cells (MCSC). Our WAP-T mouse model for oncogene-induced mammary carcinoma appeared to be suitable to test the value of ALDH1 expression as a marker for MCSC derived from WAP-T tumors. Orthotopic transplantation of isolated primary tumor cells into fat pads of syngeneic mice leads to the development of tumors of similar morphology like the primary tumor. To isolate populations enriched for MCSC we performed fluorescence activated cell sorting prior to transplantation of different amounts of ALDH1+ and ALDH1- populations. We observed tumor growth from as few as 100 cells. There was no significant difference in the morphology or growth characteristics between ALDH1+ and ALDH1- tumor cells. Similar results we obtained with a mammary carcinoma cell line established from a primary WAP-T tumor (GeTo). GeTo cells exhibit a bilinear differentiation potential with characteristics of both basal and luminal mammary epithelial cells. Upon transplantation of as few as 10 unsorted cells tumor growth can be observed. Proliferation of transplanted ALDH1- GeTo cells is only marginally delayed compared to that of transplanted ALDH+ GeTo cells.

We conclude that ALDH1 is unlikely to be a marker for a MCSC enriched population in our WAP-T mouse model. Furthermore, in this model MCSC are present at a

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much higher frequency than formerly assumed. These observations are in line with the recent publications suggesting that tumor stem cells are not necessarily a rare population.

*Supported by Deutsche Krebshilfe and by Deutsche Forschungsgemeinschaft (DFG)

Hierarchy and p53-dependent differentiation of embryonal stem/progenitor cells in mouse F9 teratocarcinoma cell line

M.-A. Kluth, R. Reimer, H. Hohenberg, W. Deppert, G.V. Tolstonog Heinrich-Pette-Institute, Hamburg, Germany

Differentiation leads to gradual specialization (commitment) of a dividing stem/progenitor cell during embryonic development and adult tissue homeostasis. Commitment accompanies the division of stem/progenitor cells and results in equal (symmetric) or unequal (asymmetrical) separation of epigenetically-determined instructions between two genetically identical daughter cells. The whole process is precisely orchestrated by regulatory factors, controlling the self-renewal and expansion of stem and progenitors on each level of commitment. Using mouse F9 teratocarcinoma cell line, a well-characterized cell differentiation model, we applied FACS-based analytical techniques to isolate and characterize the subfractions of F9 cells exhibiting properties that fit the criteria of stem/progenitor cells, namely self-renewal and potency. The “Aldefluor®”-reagent, commonly used in the stem cell research, is metabolized into a fluorescent derivate by cells expressing aldehyde dehydrogenases (ALDH), thereby allowing us to fractionate F9 cells into the ALDE+ and ALDE- populations. The microarray-based comparison of the gene expression profiles of these subpopulations with those of F9 cells treated with retinoic acid (RA) and dibutryl cAMP (dbcAMP) for differentiation and control cells revealed that ALDE+ cells are related to control, undifferentiated cells, whereas the transcriptome of ALDE- cells is similar to that of differentiated cells. As no differences in the expression of ALDH isoforms was found between ALDE+ and ALDE- populations, we analyzed the morphology and membrane potential of mitochondria, where most of the ALDH isoforms are located. Applying the fluorescent dye JC1 as a vital sensor of the mitochondrial membrane potential, we observed comparable morphology but dramatic difference in the mitochondrial activity between ALDE+ and ALDE- cells. The low membrane potential in ALDE+ cells explains the high metabolization of the “Aldefluor®”-reagent, which can passively traverse the uncharged mitochondrial membrane. ALDE+ F9 cells exhibit properties of stem/progenitor cells, as they regenerate the original F9 cell population, which retains the differentiation potential both under 2D (plastic dishes) and 3D (CarboCellTM micro-tubes) cell culture conditions. In summary, we provided evidence that the F9 cell culture comprises a heterogeneous population of stem and committed cells, which differentiate upon inductor treatment.

In order to find the regulator(s) governing the composition of F9 cell population, we paid attention to the p53 protein, which is highly expressed in ALDE+ cells, but down-regulated in ALDE- cells and upon RA/dbcAMP treatment. The p53 gene in F9 cells is wild-type and expressed at detectable levels mainly in the nucleus.

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Therefore, we performed ChIP experiments to analyze the DNA binding activity of p53 and correlate it with the activity of the respective genes. Beyond binding to promoters of known p53 target genes, like Mdm2 and Cdkn1a, we identified a new target gene transactivated by p53, Utf1, which encodes a protein known as a tran-scriptional regulator of embryonic cell differentiation. shRNA mediated knockdown of p53 resulted in an inductor-independent cell differentiation, whereas overexpression of p53 reversed this effect and protected cells against the effects of differentiation inductors. The data indicate that p53 may control the fate of stem and committed cells and regulate their asymmetric division. In support of this hypothesis, we demonstrated by BrdU pulse/chase labeling and BrdU-Hoechst 33342 quenching assays that ALDE+ cells are dividing both symmetrically and asymmetrically.

p53 as a regulator of asymmetric cell division.

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Asymmetrically dividing cells retain the BrdU label and express higher amounts of p53 than the rest of the population, providing phenomenological evidence for a role of p53 in the regulation of asymmetric division. The finding opens new perspectives for the study of p53 function in self-renewal and commitment of stem cells. Our data establish a link between loss of p53 function and initiation of tumorigenic conversion of normal stem/progenitor cell to cancer initiating cell, which lose the tight control mechanisms over cell fate regulation and expand via symmetric divisions.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6

Spontaneous F9 cell differentiation in a capillary-based 3D cell culture

M.-A. Kluth (1), R. Reimer (1), H. Hohenberg (1), S. Beck (2), M.W. Linscheid (2), W. Deppert (1), G.V. Tolstonog (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Institute of Chemistry, Humboldt-University Berlin, Germany

Cell differentiation is a fundamental process of gradual specialization (commitment) of a dividing stem/progenitor cell during embryonic development and adult tissue homeostasis. Single steps of differentiation pathway can be fol-lowed in vitro under cell culture conditions on a limited number of established cell lines which retained differentiation potential. Mouse F9 teratocarcinoma cells are widely used as a model for the analysis of the molecular mechanisms of cell differentiation and for studying steps which occur during early embryogenesis. This cell line is characterized by the inability to differentiate spontaneously (nullipotency), but upon treatment with retinoic acid (RA) alone or in combination with dibutyryl cAMP (dbcAMP), the F9 cell population differentiates towards extraembryonal parietal or visceral endoderm. Treatment with inductors induces cell arrest and partial or complete differentiation of the majority of cells, whereas a small population of cells remains in the proliferative phase. The differentiation of F9 cells is a rapid process, running into completion within 3 days of treatment. The differentiated cells are characterized by a migratory phenotype (parietal endoderm) and enhanced expression of extracellular matrix proteins, collagen IV and laminins (visceral endoderm). Inductor withdrawal by change of culture media induces rapid expansion of the proliferation-active subpopulation and the generation of differentiation-competent cells.

In order to study cell differentiation under conditions which closely mimic the natural settings, we used a unique 3D-cell culture model established in the HPI, the CarboCell TM micro-tubes. Compared to other 3D-cell culture techniques, the growth of seeded cells and the diffusion of secreted auto- and paracrine factors are restricted by the space of the capillary tube and their permeability. The transfer of cells from 2D culture into CarboCell TM micro-tubes dramatically changed the behavior of F9 cells by: (i) shortening of the dividing time from 12 hours to 3.6 hours; and (ii) spontaneous inductor-independent differentiation of F9 cells into visceral endoderm. The differentiation status of F9 cells in CarboCell TM micro-tubes was confirmed by immunohistochemical and electron microscopical detection of the components of extracellular matrix (collagen IV and α-laminin).

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Microarray-based transcriptome analysis of RA/dbcAMP-treated cells cultured under 2D conditions and cells from CarboCell TM micro-tubes revealed that around 2000 genes were similarly regulated under both cell culture conditions. Considering that RA-signaling pathway plays a key role in the differentiation of embryonic cells we tested the hypothesis that spontaneous, inductor-independent differentiation of F9 cells is triggered by retinoic acid which had been intracellularly deposited while the cells were passaged under 2D conditions in fetal calf serum (FCS) containing medium. In support of this hypothesis, we observed that pre-culturing of F9 cells in nutrient-rich, but FCS-free medium completely abolished the dif-ferentiation under 3D conditions, but did not prevent their growth. An HPLC-based analysis of the lipid fractions extracted from FCS and F9 cells cultured in the CarboCell TM micro-tubes demonstrated the presence of retinoic acid, thereby providing ultimate support for our hypothesis. In conclusion, applying CarboCell TM micro-tubes we discovered a new mechanism of embryonic cell differentiation, which is governed by storage and release of the differentiation inductor retinoic acid.

*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6

Differentiation of F9 cells under 2D and 3D cell culture.

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SPOC1 is essential for spermatogonial stem cells

H. Staege (1), S. Ficke (1,2), S. Kinkley (1), U. Matschl (1), T. Schaub (1), U. Schumacher (2), H. Will (1) (1) Heinrich-Pette Institute, Hamburg, Germany (2) University Medical Center Hamburg-Eppendorf, Dep. of Anatomy, Hamburg, Germany

We have previously reported that SPOC1-RNA levels are highest in spermatogonia, mitotic germ cells in adult testis which give rise to sperm during spermatogenesis. Using self-made mono- and polyclonal anti-SPOC1 antibodies, SPOC1 protein expression was only detected in spermatogonia, but not in other cells of the testis. The expression pattern largely overlapped with that of the PLZF protein, which is known to be essential for germ cell maintenance. The data prompted us to test whether SPOC1 plays a role in the maintenance of spermatogonia and spermatogenesis. For these studies we produced mice with doxycyclin-inducible shRNA-mediated knock-down of SPOC1 protein expression. Histological analysis of the testis obtained from doxycyclin-treated mice showed the absence of spermatogonia in some tubules and in other tubules only sertoli cells. Sertoli cells are the only cells not derived from

spermatogonia. Taken together, these data strongly suggest that the SPOC1 protein is essential and a novel marker for male germ cell generation and/or survival. These findings are consistent with additional evidences we have indicating that SPOC1 is important for cell proliferation.

*Supported by Deutsche Krebshilfe and the Müggenburg Stiftung

SPOC1 and PLZF are co-expressed in spermatogonia. Sections from adult mouse testis were co-stained with rat monoclonal anti-SPOC1 (red) and mouse monoclonal anti-PLZF (green). DNA was stained with DRAQ5.

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Activated receptor tyrosine kinases and their downstream pathways in acute myelogenous leukemia: Important targets for therapeutic intervention

S. Horn (1,2), J. Bäsecke (2), K. Schulz (1), M. Jücker (3), U. Bergholz (1), L. Trumper (2), C. Stocking (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Department of Hematology and Oncology, University of Göttingen (3) University Medical Center Hamburg-Eppendorf, Hamburg

It is now well established that leukemia, like other cancers, is the consequence of multiple genetic mutations. A popular notion is the two-hit model, in which gene mutations blocking differentiation [e.g. dysregulation of transcription factors (TF)] together with activation of a proliferation and/or anti-apoptoic signal [e.g. activated receptor tyrosine kinase (RTK)] is sufficient to induce leuekmia – and thus may represent the key events in leukemogenesis. As the latter are attractive targets for therapy, much emphasis has been placed on defining RTK mutations and their signalling pathways in leukemia. Two members of the class III RTK are frequent targets of mutations in acute myelogenous leukemia (AML). FLT3 mutations, involving either the juxtamembrane region or the tyrosine kinase domain II, are found in circa 30 % of AML samples and are not limited to a specific AML subtype. In contrast, mutations in the extracellular domain of the KIT

Activation of RTK, either by ligand or by mutation, lead to the activation of several well-characterized signaling pathways, which leads to proliferation, differentiation, and/or survival. Depicted are the major players in the PI3K-AKT pathway (orange), the JAK-STAT pathway (green), or the RAS-MAPK pathway (blue). Constitutive activation of the PI3K-AKT pathway is observed in approximately 70 % of AML samples.

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receptor are almost exclusively found in AML patients carrying translocations or inversions affecting the RUNX1 transcription or its cofactor CBFβ. Importantly, the presence of RTK mutations correlates with poor survival and/or high risk of relapse. We are interested in determining the relevant downstream signaling pathways of these mutated RTK, as well as determining if the leukemia stem cell is also “addicted” to the activated RTK for maintenance.

Mutations in RTK or RAS proteins are frequently observed in leukemia, resulting in a high frequency of constitutive activation of the PIK3K-AKT pathway. To determine if this activated pathway is sufficient to induce leukemia in mice, constitutive activated forms of the catalytic domain or localization mutants (as depicted) were tested for the tumorigenic potential by introduction via retroviral vectors into hematopoietic cells and injection into mice. As controls, empty GFP vectors, or vectors expressing constitutive activated forms of cKIT RTK are shown. Strikingly, although activated PI3K subunits are able to induce tumorigenic growth, these tumors are not as aggressive as those receiving activated KIT, which signals through multiple pathways.

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Constitutive activation of the PI3K-AKT pathway is observed in up to 70 % of acute myelogenous leukemia, primarily due to activating mutations in either receptor tyrosine kinase or RAS proteins that signal through this pathway. To investigate the relevance of an activated PI3K-AKT pathway in hematopoietic malignancies, we analyzed the effect of point mutations in the catalytic subunit of class IA PI3K (p110α), which are associated with several human neoplasms. We demonstrate that mutations in the helical (E542K, E545A) and kinase domain (H1047R) of p110α constitutively activate the PI3K-AKT pathway and lead to factor-independent growth of early hematopoietic cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not anti-apoptotic signaling of mutant p110α. In a syngenic mouse model, hematopoietic cells expressing p110α point mutations induced a leukemia-like disease characterized by anemia, neoplastic infiltration of hematopoietic organs, and 90 % mortality within 5 weeks. Our data show that an activated PI3K-AKT pathway confers factor-independency to hematopoietic cells in vitro and induces oncogenic activity in vivo, providing a useful model to evaluate pharmacological inhibitors.

The development and testing of tyrosine kinase inhibitors (TKI) specific for activated RTKs has been pushed forward during the last few years. Unfortunately, clinical trials of different TKIs have been disappointing – due to either a minimal initial response or the outgrowth of drug-resistant tumor cells. We have developed two mouse models to characterize the molecular mechanisms behind this problem. In the first mouse model, the importance of the two separate components leading to leukemia (mutated TF and activated RTK) can be independently tested. In this model, expression of the dysregulated TF (e.g. the fusion protein RUNX1/ETO) can be “turned off” by an inducible retroviral vector system, which incorporates components of the loxP-CRE recombinase system and either interferon- or tetra-cycline-responsive promoters. The activated RTK can be independently inhibited by administration of TKI. With this system, we are currently testing if the inactivation of one pathway is sufficient to reverse the leukemia. These models should provide invaluable insight into using TKI in the clinic.

*Supported by Deutsche Krebshilfe und AstraZeneca

Identification of genetic mutations that cooperate with constitutively active FLT3 in the induction of acute lymphoblastic leukemia (ALL)

A.N. Lehmann, B. Niebuhr, K. Schulz, M. Ziegler, J. Meyer, S. Horn, and C. Stocking Heinrich-Pette-Institute, Hamburg, Germany

Acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy and is a heterogeneous disease with subtypes that differ markedly in their cellular and molecular characteristics, as well as their response to therapy and subsequent risk of relapse. The FMS-like receptor tyrosine kinase FLT3 contributes to normal differentiation, proliferation and apoptosis of primitive hematopoietic

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cells in both early myeloid and lymphoid lineages. Mutations constitutively activating the FLT3 receptor are commonly found in acute leukemias of both lineages, making FLT3 one of the most frequently mutated genes in hematological malignancies. The availability of FLT3 inhibitors provides an attractive therapeutic strategy for patients harboring these mutations. In ALL, FLT3 mutations are associated with either MLL or hyperdiploid subtypes, raising the question if specific genetic events synergize with FLT3 mutations in ALL induction and if inhibiting FLT3 activation alone will be effective in leukemia treatment. To better understand the role of FLT3 activating mutations in ALL, we have established an allograft mouse model, in which bone marrow (BM) progenitors were transduced with a retroviral vector expressing FLT3 with an internal tandem duplication (FLT3-ITD), followed by transplantation into syngenic recipients. Mice receiving murine FLT3-ITD bone marrow succumbed to an aggressive leukemia with a mean latency of 45 days. Based on surface marker analysis and immunoglubulin (Ig) gene rearrangements, these tumors could be classified as pre-B-cell tumors. The disease was transplantable down to a cell number <200 and clonal (based on retroviral integration sites). Strikingly, the FLT3 inhibitor PKC412, as well inhibitors of the PI3K and MAPK pathways or dominant-negative forms of STAT5, profoundly inhibited proliferation of leukemic cells in vitro but did not induce apoptosis. However, anti-apoptotic stimulus provided in methylcellulose cultures was necessary to maintain leukemic cells in culture, which coincided with upregulation of surface expression of the Ig heavy chain of the preB-cell receptor (preBCR). These results suggest that the preBCR is active in these tumors but progression to the B-cell stage is blocked, either through FLT3 signaling and/or through independent lesions. Strikingly, each leukemia contained up to 6 to 8 independent retroviral integrations sites, a much higher number than would be expected given the circa 15-20 % infection frequencies of BM cells before transplantation. The high number of integrations could reflect two events: 1) pre-ferential infection of a subset of cells more permissive to infection; and/or 2) the preferential outgrowth of these infected cells due to the increased likelihood that cooperating genes were activated through the integration site. To determine if integration of the retroviral vector near putative oncogenes or tumor suppressors contributed to the induction of leukemia, retroviral integration sites were analyzed. Consistent with this hypothesis, integrations were found near genes that have been identified as “common integration sites” (CIS) or loci that are targeted in independent tumors – an event highly unlikely to occur merely by chance. Based on the function of gene products deregulated by the retroviral integration, we predict that disruption of the preBCR signaling pathway is necessary for FLT3-induced preB-cell leukemia.

*Supported by Deutsche Krebshilfe

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Differential regulation of human TERT gene expression and telomerase activity during liver regeneration and liver cell differentiation

H. Sirma (1), M. Kumar (1), B. Wittek (1), J. Weise (1), P. Gripon (2), S. Ande (3), K.-L. Rudolph (3), C. Günes (1). (1) Heinrich-Pette-Institute, Hamburg, Germany (2) INSERM U522, Rennes, France (3) MPI-Group, University of Ulm, Germany

Telomerase activity is non-detectable in human liver biopsies, whereas telomerase activity is upregulated during hepatocarcinogenesis, indicating that telomerase activity is repressed during differentiation of human liver and reactivation must occur during carcinogenesis. Here we analyzed the regulation of telomerase activity during liver cell proliferation and differentiation in both in vitro and in vivo models. As an in vivo model we investigated the regulation of human TERT gene promoter activity during regeneration induced by partial hepatectomy in hTERTp-lacZ trans-genic mice which contain an 8.0 kbp fragment of the human TERT gene promoter. As an in vitro model we used HepaRG cell line which recently was established as a new system for human liver cell differentiation. We found a sharp increase of human TERT promoter activity at day 3 after partial hepatectomy whereas murine TERT mRNA expression was only weakly induced at the same time. A weak but significant increase in telomerase activity was observed at day 2 after PH which did not corre-late with TERT mRNA levels during regeneration. On the other hand, telomerase activity and TERT gene expression is detectable in proliferating HepaRG cells and is markedly down-regulated as cells reach confluency and cease proliferation. Luciferase reporter gene analyses indicated that the 0.3 kbp TERT promoter fragment posseses all necessary cis regulatory elements for TERT gene repression in HepaRG cells. We established a suitable in vivo and in vitro model to dissect the molecular mecha-nisms underlying the regulation of telomerase activity in liver cells.

*Supported by Deutsche Forschungsgemeinschaft (DFG)

Regulation of human and mouse TERT promoter activity during progression of mammary carcinogenesis in WAP-T x hTERTp-lacZ bitransgenic mice

B. Wittek, M. Kumar, J. Schröder, J.M. Weise, W. Deppert, C. Günes Heinrich-Pette-Institute, Hamburg, Germany

Telomerase activity is repressed in most human somatic tissues during differentia-tion processes, but strongly up-regulated in the vast majority of human tumors. Therefore, one major focus of our research is the analysis of TERT gene expression during tumorigenesis. We have generated transgenic mice with an 8.0 kbp human TERT promoter fragment driving expression of the bacterial lacZ gene (hTERTp-lacZ transgenic mice). We could show that this transgenic mouse model provides a suitable in vivo system to analyze the expression of the human TERT gene under physiological conditions and during tumorigenesis. One model system for tumor induction and progression is the SV40 Early Region (SV40-ER) antigen induced tumor formation in the mammary tissue. A transgenic mouse model where the

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(SV40-ER) gene is under the control of the whey acidic promoter (WAP-T mice) exists in our laboratory. Here, ductal carcinomas in situ (DCIS) develop after lacta-tion in this transgenic mouse which results in invasive tumors in a defined time period. The experimentally induced DCIS is similar to the human DCIS which fur-ther develops to mammary carcinomas. Human mammary tissues, as is the case for most of the human somatic cells, are telomerase negative and reactivation of telom-erase is thought to be one of the key events during carcinogenesis, also for breast carcinomas. By back-crossing the TERT-promoter transgenic mouse with the SV40-LT transgenic mice we analyze the time point of telomerase reactivation. Our cur-rent results indicate that activation of telomerase is only detectable in invasive carci-noma but not at early time-points during tumor development. We also find that C/EBPα is a crucial repressor of TERT gene expression in normal breast and that its loss during tumorigenesis is one of the genetic events responsible for telomerase reactivation in breast tissue.

*Supported by Deutsche Krebshilfe and Erich und Gertrud Roggenbuck-Stiftung

Physiological role of cellular prion protein in insulin producing β-cells

D. J. Picketts (1), R. Reimer (2), F.W. Scott (1), A. Strom (1), A.W. Stuke (3), G.S. Wang (1) (1) Chronic Disease Program, Ottawa Health Research Institute (2) Heinrich-Pette-Institute, Hamburg, Germany (3) German Primate Centre, Göttingen

Cellular prion protein (PrPC) is ubiquitously expressed within the nervous system and is also present in several other organs and tissues. In the pancreas, PrPC is spe-cifically and abundantly expressed in the glucagon, insulin, pancreatic polypeptide, and somatostatin secreting endocrine cells which are localized in the islets of Langerhans. To date, PrPC is suggested to be involved in a number of cellular func-tions. However, the function of PrPC in the islets and specifically in insulin produc-ing ß-cells is not known. We recently reported strong PrPC expression in all hormone secreting cells in the islets. Of particular note, cytosolic PrPC aggregates were observed exclusively in insulin producing ß-cells in rats. The aim of the present study was to characterize PrPC function in insulin producing ß-cells. In order to detect PrPC interacting proteins we performed a protein overlay assay (far-Western immunoblot) using protein extracts from INS-1E rat insulinoma cells as bait and recombinant bovine PrP (rbPrP) as a probe. We detected several putative PrPC binding proteins. To date, we identified two candidate PrPC interacting molecules which were of nuclear origin. We confirmed the in vitro interaction of the rbPrP with the candidate molecules using purified recombinant proteins. Under specific immunohistochemical conditions we also detected in vivo the localization of PrPC to the nuclear lamina in endocrine and neuronal cells. Furthermore, intraperitoneal glucose tolerance tests showed that PrP0/0 mice have impaired glucoregulation. These results suggest the involvement of PrPC in epigenetic control of ß-cell function associated with glucoregulation.

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C. Molecular mechanisms for “fine tuning” normal cell processesC1. Post-transcriptional and translational control mechanismsDissecting nucleocytoplasmic transport of CD83 mRNA

J. Chemnitz (1), D. Pieper (1), B. Fries (1), C. Grüttner (1), A. Steinkasserer (2), R.H. Kehlenbach (3), J. Hauber (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) University Hospital Erlangen – Department of Dermatology, Erlangen, Germany (3) University of Göttingen – Centre of Biochemistry and Molecular Cell Biology, Göttingen, Germany

Mature dendritic cells (DC) are capable of sensitizing even naïve CD4+ and CD8+ T cells, and are therefore frequently termed “nature’s adjuvant”. Immature DC reside in the peripheral tissues and upon uptake of antigen and exposure to inflammatory stimuli, they migrate to the peripheral lymph nodes, where now fully matured, they induce antigen-specific T cell responses. The DC maturation process includes the modulation of chemokine and chemokine receptors, as well as the up-regulation of several cytokines, costimulatory and adhesion molecules.

Human CD83 serves as a major marker molecule for mature DC that belongs to the immunoglobulin super family. What is less known is that CD83 is also expressed, although to a lesser extent, by activated T and B cells and appears to be important for efficient T-cell activation.

The vast majority of cellular mRNAs are transported from the site of transcription, the cell nucleus, to the cytoplasmic site of protein synthesis via the NXF1/Tap pathway. In contrast, recently we demonstrated that the nucleocyto- plasmic transport of a small subset of cellular messages, including the CD83 mRNA, is mediated in activated T cells by the unrelated CRM1 pathway. The subsequent analysis of the CD83 transcript revealed that this mRNA contains in its coding region a highly structured cis-active RNA sequence, termed the posttran-scriptional regulatory element (PRE), and that this RNA element is directly recognized by the cel-lular RNA-binding shuttle protein HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Interestingly, PRE:HuR interaction does not affect the stability of this transcript. Instead,

Regulation of CD83 mRNA export. In the nucleus, the shuttle protein HuR binds to the PRE sequence in CD83 mRNA. Subsequently, HuR recruits its protein ligand APRIL (ANP32B) that connects the CD83 mRNA:HuR ribonucleoprotein complex and the nuclear export receptor CRM1. Nuclear export via the CRM1 pathway occurs upon phoshorylation of APRIL on residue Thr244 by casein kinase 2 (CK2).

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this interaction induces the CRM1-dependent cytoplasmic accumulation of CD83 mRNA. We also provided evidence demonstrating that the interaction of HuR and CRM1 is indirect and is accomplished by the HuR protein ligand APRIL (ANP32B).

APRIL belongs to the growing family of ANP32 proteins (ANP32a-h), a group of leucine-rich phosphoproteins, which have been linked to various cellular activities such as, for example, gene expression, signaling, adhesion and apoptosis. Our further analyses identified Thr244 to be an as yet unrecognized phosphate acceptor site in APRIL and demonstrated that phosphorylation of this site affects APRIL’s subcellular distribution. Subsequently, we identified casein kinase 2 (CK2) to be the cellular protein kinase that phosphorylates Thr244 in APRIL. We were able to show that inhibition of CK2 by a small-molecular weight inhibitor specifically abrogates CD83 de novo expression. Interestingly, this effect was caused by pronounced inhibition of the nucleocytoplasmic transport of CD83 mRNA. Thus, the pharmaceutical interference with CK2 activity may provide a novel strategy to inhibit CD83 expression, thereby blocking DC-mediated T cell activation.

*Supported in part by Wilhelm Sander-Stiftung and in part by Deutsche Forschungsgemeinschaft (DFG)

Qualitative and quantitative image analysis of ultra structural changes in sarcomere assembly of cMyBP-C knock-in mice

L. Carrier (1,2,3), T. Eschenhagen (1), B. Fraysse (2,3), J. Guiard (4), H. Hohenberg (5), B. Holstermann (5), D. Juhr (1), E. Krämer (1), G. Mearini (1), N. Mougenot (3), H. Pointu (4), R. Reimer (5), S. Schlossarek (1), K. Schwartz (2,3), M. Vernet (4), N. Vignier (2,3) (1) Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (2) Institut de Myologie, Paris, France (3) University Pierre et Marie Curie, Paris, France (4) CEA-Grenoble, Grenoble, France (5) Heinrich-Pette-Institute, Hamburg, Germany

Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein C (cMyBPC) are frequent causes of familial hypertrophic cardiomyopathy (FHC), but the mechanisms leading from mutations to disease remain elusive. The recently developed first targeted cMyBP-C knock-in mouse carries a human MYBPC3 point mutation. It is a G>A transition on the last nucleotide of exon 6, which is expected to result either in exon skipping and premature stop or production of a full-length, missense protein or both. The homozygotes exhibited myocyte and left ventricular hypertrophy with reduced fractional shortening and interstitial fibrosis, the heterozygotes developed only mild myocyte hypertrophy. In the homozygotes, the amount of missense cMyBP-C was 90 % lower and the expected truncated protein was undetectable, even after inhibiting the proteasome with MG132. Both missense and nonsense mRNAs were present, but the level of total cMyBP-C mRNA was markedly lower. Inhibition of nonsense-mediated mRNA decay in cultured cardiac

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myocytes or in vivo with cyclohex-imide or emetine increased the level of nonsense mRNA several-fold. Electron microscopy analysis has shown a correct sarcomere ultrastructure but slightly different distances between the sarcomere bands in the homozygotes. In con-clusion, these data indicate that the expression of the point mutation in the whole animal is regulated mainly at the mRNA level, involv-ing nonsense-mediated mRNA decay. These data contribute to an improved understanding of the molecular mechanisms of FHC.

Translational Elongation of Cyclin D1 requires an RNA Chaperone

J. Dittmann (1,2), G. Sommer (2), K. Reumann (1), J. Kuehnert (2), O. Mauermann (1), H. Will (1), T. Heise (1, 2) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Medical University of South Carolina (MUSC), Charleston, USA

We report that the human RNA chaperone La stimulates translation elongation of cyclin D1 by interacting specifically with the La response element (LaRE), which is located within the cyclin D1 open reading frame. By using a cell-free translation system, we showed that the presence of the LaRE hinders translation elongation and induces accumulation of a translation intermediate. Strikingly, if La protein is added to the translation reaction, the abundance of the intermediate is strongly decreased in concert with an increase in the full-length translation product. We propose a model for translational regulation of the cellular cyclin D1 mRNA pool in which the human La protein binds the LaRE located within the coding region of cyclin D1 mRNA and acts as an RNA chaperone which supports translation elongation.

*Supported by Deutsche Forschungsgemeinschaft

Ultra-structure of sarcomere assembly of cMyBP-C knock-in mice in TEM.

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The fusion protein OTT-MAL induces R-loop formation and genomic instability

G. Sommer (3), R. Fleischer (1), K. Reumann (1), M. Ruthardt (2), C. Stocking (1), H. Will (1), T. Heise (1, 3) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Johann Wolfgang Goethe-University, University Medical Center II, Hematology, Frankfurt, Germany (3) Medical University of South Carolina, Department of Biochemistry & Molecular Biology, Charleston, USA

The chromosomal translocation t(1;22) is associated with a severe childhood acute megakaryocate leukaemia (AMKL). Our study demonstrates that the corresponding fusion protein OTT-MAL interacts with endogenous RNA Polymerase II, induces transcriptional R-loop formation and phosphorylation of H2A.X (γH2A.X), a marker for genomic double strand breaks. Interestingly, the levels of spliced mRNAs are strongly reduced and OTT-MAL colocalizes with RNA polymerase II and γH2A.X in distinct nuclear foci. Our data suggest a novel oncogenic mechanism, in which a fusion protein disturbs cotranscriptional mRNA maturation and thereby induces R-loop formation, genomic instability, and eventually malignant cell transformation.

*Supported by Deutsche José Carreras Leukämie-Stiftung

Analysis of leukemia (AML-M7) associated OTT-MAL fusion protein.

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C2. Functional regulation by protein modificationsAdenovirus type 5 early region 1 B 55-kDa oncoprotein is modified by different isoforms of the small ubiquitin-related modifier (SUMO).

P. Wimmer, S. Schreiner, P. Groitl, T. Dobner Heinrich-Pette-Institute, Hamburg, Germany

We have previously shown that adenovirus type 5 (Ad5) E1B-55K is a substrate of the SUMO conjugation system in intact cells as well as in vitro. Posttranslational modification of E1B-55K by SUMO1 at lysine 104 (K104R) regulates the subcellular distribution of the viral protein in plasmid transfected and productively infected cells. Furthermore, this modification is required to completely transform primary rat cells in cooperation with Ad5 E1A and to repress p53 tumor suppressor functions. Here, we report that E1B-55K can be modified by SUMO isoforms 2 and 3 (SUMO2, SUMO3) at K104. As predicted, the molecular characteristics of SUMO2 and SUMO3 give rise to additive SUMOylation of E1B-55K generating multiple modified forms of the viral protein. We also demonstrate that E1B-55K contains three amino acid patches, which closely resemble the consensus of SUMO-interaction motifs (SIMs). This motif, V/I-X-V/I-V/I mediates non-covalent interactions with SUMO or SUMOylated proteins, and exists in nearly all proteins known to be involved in SUMO-dependent processes, such as most notably PML. Using site-directed mutagenesis we show that these motifs regulate the cellular distribution of the Ad protein and are required for efficient repression of p53-mediated transactivation in transient reporter gene assays. Future studies are directed towards elucidating the role of the SIMs in regulating protein interactions of 55K with viral and cellular factor as well as investigating their importance for E1B-55K functions in productive infection and cell transformation.

*Supported by Studienstiftung des Deutschen Volkes

The adenovirus E1B-55K oncoprotein induces SUMO modification of p53

S. Müller (1), T. Dobner (2) (1) Max Planck Institute of Biochemistry, Martinsried, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

The ability of adenovirus to induce cell transformation depends on the E1A and E1B-55K oncoproteins. While E1A functionally inactivates the retinoblastoma tumour suppressor, E1B-55K primarily interferes with the function of p53. In adenovirus transformed cells E1B-55K can directly affect p53-dependent transactivation. In virus-infected cells E1B-55K additionally cooperates with the viral E4orf6 protein to induce ubiquitin-dependent degradation of p53. Here we unravel a novel activity of E1B-55K by demonstrating that it drastically stimulates the post-translational modification of p53 by the ubiquitin-like SUMO modifier. Consistent with this finding the extent of p53 SUMOylation is highly elevated in adenovirus transformed cell lines. E1B-55Kmediated SUMOylation depends on the direct interaction of E1B-55K with p53 and additionally requires SUMO modification of E1B-55K. These data suggest that E1B-55K exploits both ubiqutin

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and ubiquitin-like systems to target host cell proteins and thus shed new light on the molecular mechanisms of E1B-55K function. Moreover, the data expand the emerging concept of dual-specificity factors that act in both the SUMO and ubiquitin pathway and identify E1B-55K as the first viral protein that shares this dual activity.

*Supported by Deutsche Forschungsgemeinschaft

Characterization of posttranslational modifications of SPOC1

T. Schaub, S. Kinkley, H. Staege, K. Reumann, H. Will Heinrich-Pette-Institute, Hamburg, Germany

SPOC1 is a 300 amino-acid protein containing a single predicted PHD (plant homeodomain) found in chromatin-associated proteins, a bipartite NLS (nuclear localization sequence) and two PEST domains (potential protein degradation motifs). Furthermore, the SPOC1 protein is predicted to be highly posttranslationally modified, suggesting that it is a very tightly regulated protein. SPOC1 contains several putative Serine and threonine phosphorylation motifs, potentially phosphorylated by Glykogen-synthase-3 β (GSK3-β), Cyclin-dependent kinases, Polo-like kinase, Casein kinase-1. In addition, there are predicted binding sites for Proline-directed kinases and Cyclins. In addition, SPOC1 contains several consensus motifs for sumoylation. To date, we have been able to show that SPOC1 is a cell-cycle regulated chromatin-associated protein and that its stability is regulated at least in part via its PEST domains and by GSK3β. In these ongoing studied we investigate the effects of the different posttranslational modifications of the SPOC1 protein on the regulation of SPOC1 functions. To this end, we have generated SPOC1 phosphorylation and sumoylation site mutants as well as phospho-specific antibodies. Elucidation of the posttranslational modification of SPOC1 will be important to understand how its subcellular localisation, interaction with cellular

SPOC1 domains and predicted posttranslational modification sites.

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proteins and various functions are regulated. In light of the accumulating data arguing that misregulated SPOC1 levels has a detrimental effect on genomic integrity, clarification of these posttranslational modifications should prove to be very informative.

*Supported by Deutsche Krebshilfe

The ubiquitin-specific protease USP7 is a target of the adenovirus E1B-55K oncoprotein

E. Koyuncu, B. Härtl, T. Dobner Heinrich-Pette-Institute, Hamburg, Germany

The 55K product from human adenovirus type 5 early region 1B (E1B-55K) belongs to a group of adenoviral regulatory proteins required for maximal virus production. In addition, E1B-55K provides functions necessary for oncogenic transformation of primary mammalian cells in culture. It is generally considered that these multifunctional properties involve a variety of physical interactions with key regulators of viral and cell growth such as E4orf6, p53, components of the DNA damage response and PML nuclear bodies as well as other still unknown factors.

The USP7 protein is relocalized to the viral transcription and replication centers during infection. A549 cells were infected with wt Ad5 at a multiplicity of 20 FFU per cell. Cells were fixed at different times after infection (h p.i.) and labeled in situ with antibodies specific for cellular PML, Ad5 E4orf3, Ad5 E1B-55K, Ad5 DBP and cellular USP7 (bottom panel), which progressively colocalizes with the DBP-positive sites of viral transcription and DNA replication during the course of the infection. Magnification x 7600.

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In a yeast two-hybrid screen, using E1B-55K as a bait, we identified the cellular protein USP7, also know as HAUSP (Herpesvirus-associated ubiquitin-specific protease), as a novel interaction partner of the 55K product. USP7 belongs to the large family of ubiquitin-specific proteases (USPs) and is a critical compo-nent of the p53-Mdm2 stress response pathway by acting as a deubiquitinase of both p53 and Mdm2. The interaction between E1B-55K and USP7 was verified by coimmunoprecipitation experiments. In vitro binding assays demonstrate that the regions involved in complex formation map to two highly conserved amino acid residues in E1B-55K and the TRAF-like domain of USP7, previously shown to mediate the binding of p53, Mdm2 and the EBNA1 protein from EBV. Furthermore, USP7 activity and binding to 55K is required for efficient transformation of primary rat cells and to maintain high levels of the viral protein indicating that USP7-mediated deubiquitination regulates E1B-55K protein stability and transforming functions. Taken together, our data identify the deubiquitinating enzyme USP7 as a novel cellular binding partner of E1B-55K and suggest an important role of USP7 for 55K protein function in virus replication and cell transformation. This together with the demonstration that EBV EBNA1 and HSV-1 ICP0 also interact with the ubiquitin-specific protease hint at the possibility that USP7 is a common target of key viral regulatory proteins.

*Supported by Wilhelm Sander-Stiftung

The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members

J. Chrystal (1), A.L. Craig (1), I. Dornreiter (2), J. Fraser (1), B. Harrison (1), T.R. Hupp (1), Y. Lin (1), M. Scott (1), N. Sphyris (1) (1) University of Edinburgh, Edinburgh, UK (2) Heinrich-Pette-Institute, Hamburg, Germany

Genetic and biochemical studies have shown that Ser(20) phosphorylation in the transactivation domain of p53 mediates p300 catalyzed DNA-dependent p53 acetylation and B-cell tumour suppression. However, the protein kinases that mediate this modification are not well-defined. A cell-free Ser(20) phosphorylation site assay was used to identify a broad range of Calcium-calmodulin kinase superfamily members including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK as Ser(20) kinases. Phosphorylation of a p53 transactivation domain fragment at Ser(20) by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbours the ubiquitin-signal for MDM2. Evaluation of these Calcium-calmodulin kinase superfamily members as candidate Ser(20) kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein depletion using siRNA can attenuate p53 phosphorylation at Ser(20), (ii) an EGFP-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in

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vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination thus controlling the specific activity of p53 as a transcription factor.

*Supported by Deutsche Forschungsgemeinschaft

Homeodomain-interacting protein kinase 2 is the ionizing radiation-activated p53 serine 46 kinase and is regulated by ATM

I. Dauth (1), J. Krüger (1,2), T.G. Hofmann (1,2) (1) German Cancer Research Center, Heidelberg, Germany (2) Heinrich-Pette-Institute, Hamburg, Germany

Phosphorylation of p53 at Ser(46) is important to activate the apoptotic program. The protein kinase that phosphorylates p53 Ser(46) in response to DNA double-strand breaks is currently unknown. The identification of this kinase is of particular interest because it may contribute to the outcome of cancer therapy. Here, we report that ionizing radiation (IR) provokes homeodomain-interacting protein kinase 2 (HIPK2) accumulation, activation, and complex formation with p53. IR-induced HIPK2 up-regulation strictly correlates with p53 Ser(46) phosphorylation. Down-regulation of HIPK2 by RNA interference specifically inhibits IR-induced phosphorylation of p53 at Ser(46). Moreover, we show that HIPK2 activation after IR is regulated by the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM). Cells from ataxia telangiectasia patients show defects in HIPK2 accumulation. Concordantly, IR-induced HIPK2 accumulation is blocked by pharmacologic inhibition of ATM. Furthermore, ATM down-regulation by RNA interference inhibited IR-induced HIPK2 accumulation, whereas checkpoint kinase 2 deficiency showed no effect. Taken together, our findings indicate that HIPK2 is the IR-activated p53 Ser(46) kinase and is regulated by ATM.

*Supported by Deutsche Krebshilfe and Deutsche Forschungsgemeinschaft

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Leibniz Center for Infection (LCI)Ultrastructural investigation of the in vitro life cycle of Schistosoma mansoni, analysed by cryo-techniques and TEM-tomography, inclusive the localisation of specific regulative proteins in situ

H. Haas (1), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1), C. Schneider (1), G. Schramm (2), M. Warmer (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Research Center Borstel, Borstel, Germany

S. mansoni affects 200 million people worldwide, often with serious consequences. This cooperation project between HPI and FZB will provide a transparent model for studying host/parasite interaction and parasite development (a.o. drug testing) and for obtaining parasite immunomodulatory key molecules. Moreover, it represents an alternative to animal experiments. The various life cycle stages of S. mansoni differ profoundly in their effects on the host. Since access to individual stages for immunobiological studies is hampered during in vivo infection, in vitro

culture of the parasite is an easy-access alternative. Moreover, only our ultrafast cryo-fixation and preparation methods can arrest the different states of infectious eggs. Preserving the life-like state of the parasite we are able to study the time course of expression of individual immuno-modulatory egg molecules by 3D ultrastructural investigations combined with the pinpoint localisation of these molecules by immunocytological studies.

Ultrastructure of an Schistosoma mansoni egg after high pressure freezing in situ and cryoprocessing in the transmission electron microscope.

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Electron microscopical dissection of the exoerythrocytic forms of Plasmodium berghei in situ

V.T. Heussler (2), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1), A. Rennenberg (2) C. Schneider (1) (1) Heinrich-Pette-Institute, Hamburg, Germany (2) Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

The initiation of clinical malaria evolves after the schizogonic division of the exoerythrocytic forms (EEFs) of the parasite and the formation of merozoites inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes still remains unknown. Recently it was shown that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as “eat me” signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from the host immune system. With the help of specially adapted correlative light / electron microscopy methods and novel pre-selection and micro-preparation techniques we were able to localize and to make a pin-point dissection of EEF´s in situ in the liver.

A: Hepatic merozoit of Plamodium berghei in liver tissue (arrow). B: Schematic drawing of the different budding steps of parasite-filled vesicles (merosomes) into the sinusoid lumen (from Sturm et al., 2006)

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Personelle Zusammensetzung der Wissenschaftlichen Abteilungen, Forschungs- und Nachwuchsgruppen

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Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

Bachelor Studenten

Abteilung Molekulare Virologie

Thomas Dobner, Prof. Dr. rer. nat.

Barbara Härtl, Dr. rer. nat.Kathrin Kindsmüller, Dr. rer. nat.*Timo Sieber, Dr . rer. nat.

Willhelm Ching*, Dipl. Chem.Orkide Dirlik-Koyuncu, M.Sc. Natascha Kömm*, Dipl. Biol.Emre Koyuncu, M.Sc.Agnes Lesniewski*, Dipl. Biol.Daniela Müller*, Dipl. Biol.Marie-Sophie Sander*, Cand. med.Timo Sieber*, Dipl. Biol.Melanie Schmid*, Dipl. Biol. Sabrina Schreiner, Dipl. Biol.Thomas Speiseder, Dipl. Biol.Peter Wimmer*, Dipl. Biol.

Wilhelm Ching*Franziska KoppFlorian LeinenkugelAndreas MalitzkyChristian Peters*Melanie Schmid*Peter Wimmer*

Julia GodauMaria SekutoviczTimo Fischer

146

Personelle Zusammensetzung

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147

Personelle Zusammensetzung

Karolin Berg*Wilhelm Ching*Anabelle von Davier*Nicholas Dobner*Christine Henning*Florian Henkel*Hannes Homfeld*Linda Hüske*Fabian Imse*Franziska Kopp*Denise Lehnhardt*Agnes Lesniewski*Kristina MetzSandra Postels*Folusho Osuntuyi*Kristina Ritter*David Rohde*Mareike RuhnauPeggy Synwoldt*Anne Winkler*

Gabriele Dobner*Peter Groitl, Dipl. Biol.

Anna Bresovitsky*Peter Forster*Martin KruseTimo Sieber*Susan WatsonPeter Wimmer*Tina Wunder*Harald Wodrich

Edda Renz

Franz Rinninger, Prof. Dr. med.May Brundert, PD Dr. med.Udo Schumacher, Prof. Dr. med.Olaf Pongs, Prof. Dr. med.

(*zeitweise beschäftigt)

Praktikanten

Technische Assistenten

Gäste

Sekretariat

Assoziierte KlinischeKooperationsgruppen

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Personelle Zusammensetzung

Hans Will, Prof. Dr. rer. nat.

Anneke Funk*, Dr. rer. nat.Gabor Rohaly, Dr. rer. nat.Gunhild Sommer*, Dr. rer. nat.Hannah Staege, Dr. rer. nat.Hüseyin Sirma, PD Dr. med.Tilman Heise*, Dr. rer. nat.Irena Dornreiter, Dr. rer. nat.Mouna Mhamdi*, Dr. rer. nat.Sarah Kinkley*, Dr. rer. nat.

Alejandro Mena-Nunez*, Dipl. Biol.Andreas Mund*, Dipl. Biol.Cynthia Olotu*, cand. med.Sarah Kinkley*, Dipl. biol.Malte Kriegs*, Dipl. Biol.Mouna Mhamdi*, Dipl. Biol.Stefan Ficke*, cand med.

Marc Bohlmann*Katrin Gießelmann*Christine Kranig*Theres Schaub*

Abteilungsleiter

Wissenschaftler

Doktoranden

Diplomanden

148

Abteilung Allgemeine Virologie

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Personelle Zusammensetzung

Katharina Korf*Verena Deing*Tobias Schubert*

Heithem Boukari*Lutz Homann*

Agnes Hunger* Annette Preuß*Daniela Ploen* Deborah Maus*Heithem Boukari* Lutz Homann*Katharina Korf* Katja Kleinsteuber*Maren Ahlers* Melanie Piedavent*Nancy Lachner* Sabrina Köcher*Sandra Münch* Tina Bohstedt*Tobias Schubert* Verena Deing *Theres Schaub* Anneli Sagar*Emily-Marie Latuske* Nora Pallmann*Margarethe Winnicki* Leah Eissing*Katrin Wagner Prigge*

Heike Hildebrandt*Kerstin ReumannNicole Lohrengel*Oliver Mauermann*Susann Schönefeldt*Silke Dehde* Urte Matschl*

Prof. U. Schumacher*

Ingrid Michalski*Jacqueline Bukatz*

(*zeitweise beschäftigt)

Diplomanden

Bachelor Studenten

Praktikanten

Technische Assistenten

Gäste

Sekretariat

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Wolfgang Deppert, Prof. Dr. rer. nat.

Wolfgang Bohn, PD Dr. rer. nat.Michael Bruns, PD Dr. med. vet.Cagatay Günes, PD Dr. rer. nat.Christina Heinlein, Dr. rer. nat.*Heike Helmbold, Dr. rer. nat.*Andrea Hermannstädter, Dr. rer. nat.*Frauke Krepulat, Dr. rer. nat.*Mukesh Kumar, Dr. rer. nat.*Claudia Mänz, Dr. rer. nat.*Annette März, Dr. rer. nat.*Lars Tögel, Dr. rer. nat.*Genrich Tolstonog, Dr. rer. nat.Julia Weise, Dr. rer. nat.*Britta Wittek, Dr. rer. nat.*

Satish Babu, Master Sci.Heidrun Gevensleben, cand. med.*Janosch Hildebrand, Dipl. Biol.*Mike Jahn, Dipl. Biol.*Andreas Kluth, Master Sci.*Tanja Knauer, Dipl. Biol.*Eva Lenfert, Dipl. Biol.*Timo Quante, Dipl. Biochem.Martin Rütze, Dipl. Biotech.Claudia Schlesiger, Dipl. Biol.*Jara Wanger, cand. med. vet.*Florian Wegwitz, Dipl. Biochem.

Abteilungsleiter

Wissenschaftler

Doktoranden

150

Personelle Zusammensetzung

Abteilung Tumorvirologie

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Katharina Gruner*Norbert Günther*Natascha Kömm*Dorothea Pieper*Jarob Saker*

Anneli Sagar*Thomas Schulze*

Andrea DiesterbeckSilvia Dolski*Marion KühlChristine Loscher*Jasmin Oehlmann*Gundula Pilnitz-Stolze*Annette Preuß*Julia SchröderGabriele WarneckeDoris Weidemann

Claudia Annan*Marie Brazdova*, Dr. rer. nat.Umberto Galderisi*, Prof. Dr.Kamila Iwanski, cand. med.*Ella Kim, PD Dr. rer. nat.Katharina Möllmann*Peter Pably, Dipl. Biochem.*Klaus Wiegers, Dr. rer. nat.*M. Cengiz Yakicier, Dr. rer. nat.*

Irmgard Haye*Monika Müller*Semsa Reus

Martina Hintz-Malchow

(*zeitweise beschäftigt)

Diplomanden

Praktikanten

TechnischeAssistenten

Gäste

Spülküche

Sekretariat

151

Personelle Zusammensetzung

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Joachim Hauber, Prof. Dr. rer. nat.

Jan Chemnitz, Dr. rer. nat.Janet Chusainow, Dr. rer. nat.*Ilona Hauber, Dr. rer. nat.Helga Hofmann-Sieber, Dr. rer. nat.*Claus-Henning Nagel, Dr. rer. nat.*Poornima Priyadarshini C.G., Dr. rer. nat.*Klaus Wiegers, Dr. med.*

Christina Ehlers, Dipl. Chem.*Dirk Hoffmann, Dipl. Biochem.Marcel Krepstakies, Dipl. Chem.Dorothea Pieper, Dipl. Biol.*

Stephan Hofmann*

Stephan Hofmann*Susanne Scheu*

Abteilungsleiter

Wissenschaftler

Doktoranden

Bachelor Studenten

Praktikanten

152

Personelle Zusammensetzung

Abteilung Zellbiologie und Virologie

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Personelle Zusammensetzung

TechnischeAssistenten

Gäste

Sekretariat

Assoziierte KlinischeKooperationsgruppe

Bettina AbelNiklas Beschorner, Dipl. Biotech.*Andrea BunkCordula GrüttnerHendrik Plagmann*Britta Weseloh

Basma Abdel Motaal*Dr. Gerald Bacher*Stephanie Frankenberger*Nele Jensen*Prof. Dr. Annette Kaiser*

Ute Neumann

Kristina Colberg *Philip Hartjen, Dr. rer. nat.*Nele Jensen*Silke Kummer*Janine Mohn*Dorothea Pieper*Julian Schulze zur Wiesch, Dr. med.*Ingrid Stahmer, Dr. rer. nat.*Adriana Thomssen*Jan van Lunzen, PD Dr. med.

(*zeitweise beschäftigt)

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Personelle Zusammensetzung

Heinrich Hohenberg, Dr. rer. nat.

Rudolph Reimer, Dr. rer. nat.

Robert Getzieh* Yonas Shaikh*

Hendrick Herrmann*Rasa Raithore*

Carola SchneiderBarbara HolstermannMartin WarmerHendrick Herrmann*

(*zeitweise beschäftigt)

Forschungsgruppenleiter

Wissenschaftler

Diplomanden

Bachelor Studenten

Praktikanten

TechnischeAssistenten

Forschungsgruppe Elektronenmikroskopie und Mikrotechnologie

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Personelle Zusammensetzung

Carol Stocking, Dr. phil.

Stefan Horn, Dr. rer. nat.Maike Täger, Dr. rer. nat.

Folake Akinduro, Dipl. Biol.Anneke Lehmann, Dipl. Biol.Birte Niebuhr, Dipl. Agarbiol.Andrea Schüler, Dipl. Biol.Katrin Schulz, Dipl. Biochem.

Kira Behrens* Sabine Leisten*Kira Behrens*Frederik Tellkamp*

Ulla BergholzUrsula Müller, Dipl.-Ing.Susanne RoscherMarion Ziegler

Klaus Harbers, Dr. rer. nat.Jürgen Löhler, Dr. med.Vladimir Prassolov, Prof. Dr

(*zeitweise beschäftigt)

Forschungsgruppenleiterin

Wissenschaftler

Doktoranden

Diplomanden

Praktikanten

TechnischeAssistenten

Gäste

Forschungsgruppe Molekulare Pathologie

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Personelle Zusammensetzung

Adam Grundhoff, Dr. rer. nat.

Thomas ChristallaNicole WalzThomas GüntherChristine Henning

Uwe Tessmer, Dipl. Ing.

Dr. Nicole Fischer (UKE)

(*zeitweise beschäftigt)

Nachwuchsgruppe Zelluläre Virusabwehr

Nachwuchsgruppenleiter

Doktoranden

Technische Assistenten

Gäste

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Personelle Zusammensetzung

Nachwuchsgruppe Virus-Pathogenese

Michael Schindler, Dr. biol. hum.

Carina BanningSwantje GundlachMiriam Widder

Nina Eickel*Heike Ströver*

Karen Bayer*Nina Eickel*Konstantin Hoffer*Heike Ströver*

Alicja Iwanski, Dipl. Biol.

(*zeitweise beschäftigt)

Nachwuchsgruppenleiter

Doktoranden

Diplomanden

Praktikanten

TechnischeAssistenten

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2007

Peer-Reviewed Original Publications

Balabanov, S., Gontarewicz, A., Ziegler, P., Hartmann, U., Kammer, W., Copland, M., Brassat, U., Priemer, M., Hauber, I., Wilhelm, T., Schwarz, G., Kanz, L., Bokemeyer, C., Hauber, J., Holyoake, T.L., Nordheim, A., and Brümmendorf, T.H. (2007). Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach. Blood 109, 1701-1711.

Baschuk, N., Utermöhlen, O., Gugel, R., Warnecke, G., Karow, U., Paulsen, D., Brombacher, F., Krönke, M., and Deppert, W. (2007). Interleukin-4 Impairs Granzyme-Mediated Cytotoxity of Simian Virus 40 Large Tumor Antigen-Specific CTL in BALB/c Mice. Cancer Immunol. Immunother. 56, 1625-1636.

Bruns, K., Studtrucker, N., Sharma, A., Fossen, T., Mitzner, D., Eissmann, A., Tessmer, U., Röder, R., Henklein, P., Wray, V., Schubert, U. (2007). Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein. J Biol. Chem. 5; 282: 353-363.

Cammenga, J., Niebuhr, B., Horn, S., Bergholz, U., Putz, G., Buchholz, F., Löhler, J., and Stocking, C. (2007). RUNX1 DNA-binding mutants, associated with minimally differentiated acute myeloid leukemia, disrupt myeloid differentia-tion. Cancer Res. 67, 537-545.

Craig, A.L., Chrystal, J., Fraser, J., Sphyris, N., Lin, Y., Harrison, B., Scott, M., Dornreiter, I., and Hupp, T.R. (2007). The MDM2 Ubiquitination Signal in the DNA-Binding Domain of p53 Forms a Docking Site for Calcium Calmodulin Kinase Superfamily Members. Mol. Cell. Biol. 27, 3542-55.

Dauth, I., Krüger, J., and Hofmann, T.G. (2007). Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation-Activated p53 Serine 46 Kinase and Is Regulated by ATM. Cancer Res. 67, 2274-2279.

Deppert, W. (2007). Mutant p53: From Guardian to Fallen Angel? Oncogene 26, 2142-2144. Review.

Franke, C., Matschl, U., and Bruns, M. (2007). Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfec-tious subviral particles. Virology 359, 126-136

Fries, B., Heukeshoven, J., Hauber, I., Grüttner, C., Stocking, C., Kehlenbach, R.H., Hauber, J., and Chemnitz, J. (2007). Analysis of nucleocytoplasmic traf-ficking of the HuR ligand APRIL and its influence on CD83 expression. J. Biol. Chem. 282, 4504-4515.

Funk, A., Mhamdi, M., Will, H., and Sirma, H. (2007). Avian hepatitis B viruses: Molecular and cellular biology, phylogenesis, and host tropism. World J. Gastroenterol 13, 91-103.

Veröffentlichungen

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Veröffentlichungen

Kaiser, A., Hammels, I., Gottwald, A., Nassar, M., Zaghloul, M.S., Motaal, B.A., Hauber, J., and Hoerauf, A. (2007). Modification of eukaryotic initiation fac-tor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties. Bioorg. Med. Chem. 15, 6200-6207.

Kim, E., and Deppert, W. (2007). Interactions of Mutant p53 with DNA: Guilt by Association. Oncogene 26, 2185-2190. Review.

Kindsmuller, K., Groitl, P., Hartl, B., Blanchette, P., Hauber, J., and Dobner, T. (2007). Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation. Proc Natl Acad Sci USA 104, 6684-6689.

Krieghoff, E., Milovic-Holm, K., and Hofmann, T.G. (2007). FLASH meets nuclear bodies: CD95 receptor signals via a nuclear pathway. Cell Cycle 6, 771-775.

Krummheuer, J., Johnson, A.T., Hauber, I., Kammler, S., Anderson, J.L., Hauber, J., Purcell, D.F., and Schaal, H. (2007). A minimal uORF within the HIV-1 vpu leader allows efficient translation initiation at the downstream env AUG. Virology 363, 261-271.

Maenz,C., Chang,S.-F., Iwanski,A., and Bruns,M. (2007). Entry of duck hepatitis B virus into primary duck liver and kidney cells after discovery of a fusogenic region within the large surface protein. J. Virol. 81, 5014-5023.

Matthes, E., Funk, A., Krahn, I., Gaertner, K., von Janta-Lipinski, M., Lin, L., Will, H., and Sirma, H. (2007). Strong and selective inhibitors of HBV-replication among novel N4-hydroxy-and 5-Methyl-β-L-deoxycytidine ana-logues. Antimicrobial Agents and Chemotherapy 51, 2523-2530.

Metzner, A. Horstmann, M.A, Fehse, B., Ortmeyer, G., Niemeyer, C.M., Stocking, C., Mayr, G.W. and Jücker, M. (2007). Gene transfer of SHIP-1 inhibits prolife-ration of juvenile myelomonocytic leukemia cells carrying KRAS2 or PTPN11 mutations. Gene Therapy 14, 699-703.

Mhamdi, M., Funk, A., Hohenberg, H., Will, H., and Sirma, H. (2007). Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles. Hepatology. 46, 95-106.

Milovic-Holm, K., Krieghoff, E., Jensen, K., Will, H., and Hofmann, T.G. (2007). FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies. EMBO J 26, 391-401.

Münch J, Rajan D, Schindler M, Specht A, Rücker E, Novembre FJ, Nerrienet E, Müller-Trutwin MC, Peeters M, Hahn BH, Kirchhoff F. (*Equal contribution) (2007). Nef-mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication are Fundamental Properties of Primate Lentiviruses. J Virol 81, 13852-13864.

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Veröffentlichungen

Münch J, Rücker E, Ständker L, Adermann K, Goffinet C, Schindler M, Wildum S, Chinnadurai R, Rajan D, Specht A, Giménez-Gallego G, Sánchez PC, Fowler DM, Koulov A, Kelly JW, Mothes W, Grivel JC, Margolis L, Keppler OT, Forssmann WG, Kirchhoff F. (2007). Semen-Derived Amyloid Fibrils Drastically Enhance HIV Infection. Cell. 131, 1059-1071.

Niendorf, S., Oksche, A., Kisser, A., Löhler, J., Prinz, M., Schorle, H., Feller, S., Lewitzky, M., Horak, I., Knobeloch, K.P. (2007). Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo. Mol. Cell. Biol. 27, 5029-5039.

Reifenberg, K., Lehr, H.A., Torzewski, M., Steige, G., Wiese, E., Küpper, I., Becker, C., Ott, S., Nusser, P., Yamamura, K., Rechtsteiner, G., Warger, T., Pautz, A., Kleinert, H., Schmidt, A., Pieske, B., Wenzel, P., Münzel, T., Löhler, J. (2007) Interferon-gamma induces chronic active myocarditis and cardiomyopathy in transgenic mice. Am. J. Pathol. 171, 463-472.

Rodenburg, M. Fischer, M., Engelmann, A., Ogryzlo-Harbers, S., Ziegler, M., Löhler, J. and Stocking, C. (2007). Importance of receptor usage, Fli1 activation, and mouse strain in the “stem-cell” specificity of 10A1-MuLV leukemogenicity. J. Virol. 81, 732-742.

Sarkar, I., Hauber, I., Hauber, J., and Buchholz, F. (2007). HIV-1 proviral DNA excision using an evolved recombinase. Science 316, 1912-1915.

Schindler M, Rajan D, Specht A, Ritter C, Pulkkinen K, Saksela K, Kirchhoff F. (2007). Association of Nef with p21-Activated Kinase 2 Is Dispensable for Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity in Ex Vivo-Infected Human Lymphoid Tissue. J Virol 81, 13005-13014.

Sieber, T., and Dobner, T. (2007). Adenovirus type 5 early region 1B 156R protein promotes cell transformation independently of repression of p53-stimulated transcription. J Virol 81, 95-105.

Sirma, H., Funk, A., Gerlich, W., and Schildgen, O. (2007). Does pre-treatment with lamivudine prime for adefovir resistance of HBV infection? J. Antimicrobial Chemotherapy 60, 448-449.

Strom, A., Wang, G.S., Reimer, R., Finegood, D.T., Scott, F.W. (2007). Pronounced cytosolic aggregation of cellular prion protein in pancreatic beta-cells in response to hyperglycemia. Lab Invest. 87, 139-149.

Tromsdorf, U.I., Bigall, N.C., Kaul, M.G., Bruns, O.T., Nikolic, M.S., Mollwitz, B., Sperling, R.A., Reimer, R., Hohenberg, H., Parak, W.J., Förster, S., Beisiegel, U., Adam, G., Weller, H. (2007). Size and surface effects on the MRI relaxivity of manganese ferrite nanoparticle contrast agents. Nano Lett. 7, 2422-2427.

van Niekerk, EA., Willis, D.E., Chang, J.H., Reumann, K., Heise, T., and Twiss, J.L. (2007). Sumoylation in axons triggers retrograde transport of the RNA binding protein La. Proc. Natl. Acad. Sci. USA 104, 12913-12918.

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Non Reviewed Publications, Reviews and Book Chapters

Bruns, M., and Maenz, C. (2007). Requirement of activation for hepatitis B virus infection. Nature Precedings: http://hdl.nature.com/10101/npre.2007.221.1.

Groitl, P., and Dobner, T. (2007). Construction of adenovirus type 5 early region 1 and 4 virus mutants. In: Adenovirus Methods and Protocols, W.S. Wold, and A.E. Tollefson, eds. (Totowa, NJ, Humana Press Inc.), pp. 29-39.

Nevels, M., and Dobner, T. (2007). Determination of the transforming activities of adenovirus oncogenes. In: Adenovirus Methods and Protocols, W.S. Wold, and A.E. Tollefson, eds. (Totowa, NJ, Humana Press Inc.), pp. 187-195.

Prassolov, V., Hein, S., Ivanov, D., Lohler, J., Spirin, P., and Stocking, C. (2007). The M813 retrovirus belongs to a unique interference group and is highly fusogenic, In Stem Cells and their Application for Clinical Therapy, B. Fehse, N. Bilko, C. Stocking, W. Ostertag, and A. Zander, eds. (Dordrecht: Springer), pp.223-244.

Reimer, R., Warmer, M., Franz, N., Holstermann B., Hohenberg H. (2007) An ImageJ Plugin for Stereoscopic Visualization of Three Dimensional Structures on Images Obtained by Environmental Scanning Electron Microscopy (ESEM). Microscopy and Microanalysis 13 (Suppl. 02), pp. 1676-1677.

Reimer, R., Warmer, M., Holstermann, B., Hohenberg, H. (2007). Strategies for the Fine-Morphological Analysis of Fully Hydrated and Life-Like Preserved Biological and Clinical Material in the SEM. Microscopy and Microanalysis 13 (Suppl. 02), pp. 1458-145.

Stocking, C., Niebuhr, B., Fischer, M., Täger, M., and Cammenga, J. (2007). The RUNX1 transcription factor: A gatekeeper of acute leukemia, In Stem Cells and their Potential for Clinical Application, B. Fehse, N. Bilko, C. Stocking, W. Ostertag, and A. Zander, eds. (Dordrecht: Springer), pp. 183-199.

Sullivan CS, Grundhoff A. (2007). Identification of viral microRNAs. Methods Enzymol 427, 3-23.

van Lunzen, J. (2007). How will CCR5 antagonists influence the recommendations for the antiretroviral tratment of HIV-1 infection. Eur. J. Med. Res. 12, 435-440.

van Lunzen, J., and Hoffmann,C. (2007). Virological rebound and ist conse-quences during treatment interruption. Cur. Opin. HIV and AIDS 2, 1-5.

van Lunzen, J. (2007). Treatment of heavily antiretroviral-experienced HIV-infected patients. AIDS Rev. 9, 246-253.

Wepf, R., Droste, M.S., Schertel, A. and Hohenberg H. (2007). Zoom-in beyond light microscopy: A new approach for biological structure research – correlati-ve light and electron microscopy on one and the same sample” 12/2007 G.I.T. Imaging & Microscopy, Spec. Ed. “Advances in Microscopy”.

Wepf, R., Richter, T., Biel, S., Schlüter, H., Fischer, F., Wittern, K.P., Hohenberg, H. (2007). Multimodal imaging of skin structures: Imagining imaging of the Skin. In: Bioengineering of the skin: Skin Imaging and Analysis 2nd Ed. 2007, Informa Healthcare USA, New York.

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2008

Peer-Reviewed Original Publications

Asang, C., Hauber, I., and Schaal, H. (2008). Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer. Nucleic Acids Res. 36, 1450-1463.

Blackford, A.N., Bruton, R.K., Dirlik, O., Stewart, G.S., Taylor, A.M., Dobner, T., Grand, R.J., and Turnell, A.S. (2008). A role for E1B-AP5 in ATR signaling pathways during adenovirus infection. J Virol 82, 7640-7652.

Blanchette, P., Kindsmüller, K., Groitl, P., Dallaire, F., Speiseder, T., Branton, P.E., and Dobner, T. (2008). Control of mRNA export by adenovirus E4orf6 and E1B55K proteins during productive infection requires the E4orf6 ubiquitin ligase activity. J Virol 82, 2642-2651.

Chemnitz, J., Pieper, D., Grüttner, C., and Hauber, J. (2008). Phosphorylation of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression. Eur. J. Immunol. 39, 267-79.

Deppert, W. (2008). SIRT1 Protein Levels in Cancer: Tuning SIRT1 to the Needs of a Cancer Cell. Cell Cycle 7, 2947-2948.

Engelmann, A., Speidel, D., Bornkamm, G.W., Deppert, W., and Stocking, C. (2008). Gadd45ß is a Pro-Survival Factor Associated with Stress-Resistant Tumors. Oncogene 27, 1429-1438.

Everett, R.D., Parada, C., Gripon, P., Sirma, H., and Orr, A. (2008). Replication of ICP0-null-mutant herpes simplex virus type 1 Is restricted by both PML and Sp100. J. Virol. 82, 2661-2672.

Funk, A., Mhamdi, M., Hohenberg, H., Heeren, J., Reimer, R., Lambert, C., Prange, R., and Sirma, H. (2008) Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry. J Virol. 82, 10532-10542.

Goebel, T., Ulmer, D., Projahn, H., Kloeckner, J., Heller, E., Glaser, M., Ponte-Sucre, A., Specht, S., Sarite, S.R., Hoerauf, A., Kaiser, A., Hauber, I., Hauber, J., and Holzgrabe, U. (2008). In search of novel agents for therapy of tropical diseases and human immunodeficiency virus. J. Med. Chem. 51, 238-250.

Härtl, B., Zeller, T., Blanchette, P., Kremmer, E., and Dobner, T. (2008). Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription. Oncogene 27, 3673-3684.

Heinlein, C., Krepulat, F., Löhler, J., Speidel, D., Deppert, W., and Tolstonog, G. (2008). Mutant p53R20H Gain of Function Phenotype in a Mouse Model for Oncogene-Induced Mammary Carcinogenesis. Int. J. Cancer 122, 1701-1709.

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Veröffentlichungen

Henning, K., Heering, J., Schwanbeck, R., Schroeder, T., Helmbold, H, Schäfer, H., Deppert, W., Kim, E., and Just, U. (2008) Notch1 Activation Reduces Proliferation in the Multipotent Hematopoietic Progenitor Cell Line FDCP-mix through a p53-Dependent Pathway but Notch1 Effects on Myeloid and Erythroid Differentiation are Independent of p53. Cell Death Differ. 15, 298-407.

Horn, S., Bergholz, U., Jücker, M., McCubrey, J.A., Trümper, L. Stocking, C., and Bäsecke, J.: (2008). Oncogenic function of mutations in the catalytic subunit of PI3K in hematopoietic cells. Oncogene 27, 4096-4106.

Hoxha,E., Fehse B., Ortmeyer,G. Stocking,C., Mayr,G.W. and Jücker,M. (2008). Overexpression of the p85α regulatory subunit of phosphatidylinositol 3-kinase inhibits GM-CSF-dependent colony formation of CD34+ hematopoietic proge-nitor cells. Leuk. Lymphoma 22, 1-3.

Hutten, S., Wälde, S., Spillner, C., Hauber, J., and Kehlenbach, R.H. (2008). The nuclear pore component Nup358 promotes transportin-dependent nuclear import. J. Cell Sci., in press.

Kawamata, N., Ogawa, S., Zimmermann, M., Niebuhr, B., Stocking, C., Sanada, M., Hemminki, K., Yamatomo, Nannya, Y., Koehler, R., Flohr, T., Miller, C.W., Harbott, J., Ludwig, W.-D., Stanulla, M., Schrappe, M., Bartram, C.R., and Koeffler, H.P. (2008). Cloning of genes involved in chromosomal translocations by high resolution single nucleotide polymorphism genomic microarray. Proc. Natl. Acad. Sci. USA 105, 11921-6.

Kirchhoff F, Schindler M, Specht A, Arhel N, Münch J. (2008). Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci. 65, 2621-2636.

Krack, M., Hohenberg, H., Kornowski, A., Lindner, P., Weller, H., Förster, S. (2008) Nanoparticle-loaded magnetophoretic vesicles. J Am Chem Soc. 130, 7315-7320.

Lehmann, C., Harper, J.M., Taubert, D., Hartmann, P., Fätkenheuer, G., Jung,N., van Lunzen, J., Stellbrink, H.J., Gallo, R.C., and Romerio, F. (2008). Increased interferon alpha expression in circulating plasmacytoid dendritic cells of HIV-1-infected patients. J. Acquir. Immune Defic. Syndr. 48, 522-530.

Metzner, A., Precht, C., Fehse, B., Fiedler, W., Stocking, C., Günther, A., Mayr, G.W. and Jücker, M. (2008) Reduced proliferation of CD34+ cells from patients with acute myeloid leukemia after gene transfer of INPP5D. Gene Therapy, 16, 570-573

Maenz, C., Loscher, C., Iwanski, A., and Bruns, M. (2008). Inhibition of duck hepatitis B virus infection of liver cells by combined treatment with viral e anti-gen and carbohydrates. J. Gen. Virol. 89, 313-326.

Miller, A.D., Bergholz, U., Ziegler, M, and Stocking, C. (2008). Identification of the myelin protein plasmolipin as the cell entry receptor for Mus caroli endoge-nous retrovirus. J. Virol. 82, 6862-6868.

Muller, S., and Dobner, T. (2008). The adenovirus E1B-55K oncoprotein induces SUMO modification of p53. Cell Cycle 7, 754-758.

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Veröffentlichungen

Netter, H.-J., Chang, S.-F., and Bruns, M. (2008). Host-range and pathogenicity of hepatitis B virus. Future Virol. 3, 83-94.

Niebuhr, B., Fischer, M. Täger, M., Cammenga, J., and Stocking, C. (2008). Gatekeeper function of the RUNX1 transcription factor in acute leukemia. Blood Cells Mol. Dis. 40, 211-218.

Niebuhr, N., Iwanski, G.B., Schwieger, M., Roscher, S., Stocking C., Cammenga, J. (2008). Investigation of C/EBPα function in human (versus murine) mye-lopoiesis provides novel insight into the impact of CEBPA mutations in acute myelogenous leukemia (AML). Leukemia 23, 978-983

Pfuhl, T., Mamiani, A., Durr, M., Welter, S., Stieber, J., Ankara, J., Liss, M., Dobner, T., Schmitt, A., Falkai, P., et al. (2008). The LARK/RBM4a protein is highly expressed in cerebellum as compared to cerebrum. Neurosci Lett 444, 11-15.

Raimondo, G., Allain, JP., Brunetto, MR., Buendia, MA., Chen, DS., Colombo, M., Craxì, A., Donato, F., Ferrari, C., Gaeta, GB., Gerlich, WH., Levrero, M., Locarnini, S., Michalak, T., Mondelli, MU., Pawlotsky, JM., Pollicino, T., Prati, D., Puoti, M., Samuel, D., Shouval, D., Smedile, A., Squadrito, G., Trépo, C., Villa, E., Will, H., Zanetti, AR., and Zoulim, F. (2008). Statements from the Taormina expert meeting on occult hepatitis B virus infection. J. Hepatol. 49, 652-657.

Ruetze, M., Gallinat, S., Lim, I.J., Chow, E., Phan, T.T., Staeb, F., Wenck, H., Deppert, W, and Knott, A. (2008). Common Features of Umbilical Cord Epithelial Cells and Epidermal Keratinocytes. J. Dermatol. Sci. 50, 227-231.

Schindler M, Schmökel J, Specht A, Li H, Münch J, Khalod M, Sodora DL, Hahn BH, Silvestri G, Kirchhoff F. (2008). Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4+ T cells in natural SIV infection. PLoS Pathog 4(7): e1000107. doi: 10.1371/journal.ppat. 1000107.

Schüler, A., Schwieger, M., Engelmann, A., Weber, K., Horn, S., Müller, U., Arnold, M., Olson, E. and Stocking, C. (2008). The MADS transcription factor Mef2c is a pivotal modulator of myeloid cell fate. Blood 111, 4532-4541.

Schwalbe, M., Ohlenschläger, O., Marchanka, A., Ramachandran, R., Häfner, S., Heise, T. and Görlach, M. (2008). Solution structure of the stem-loop of the hepatitis B virus post-transcriptional regulatory element. Nucl. Acids Res. 36, 1681-1689.

Specht A, Degottardi MQ, Schindler M, Hahn B, Evans DT, Kirchhoff F. (2008). Selective downmodulation of HLA-A and -B by Nef alleles from different groups of primate lentiviruses. Virology 373: 229-237.

Specht, S., Sarite, S.R., Hauber, I., Hauber, J., Görbig, U.F., Meier, C., Bevec, D., Hoerauf, A., and Kaiser, A. (2008). The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytoki-ne release and parasitic deoxyhypusine synthase. Parasitol. Res. 102, 1177-1184.

Toussaint H, Gobert FX, Schindler M, Banning C, Kozik Patrycja, Jouve M, Kirchhoff F, Benaroch P. (2008). HIV-1 Nef expression prevents AP-2-mediated internalization of the MHC II-associated invariant chain. J Virol 82, 8373-8782.

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Veröffentlichungen

Weber, K., Bartsch, U., Stocking, C., Fehse, B. (2008). A multicolor panel of novel lentiviral "Gene Ontology" (LeGO) vectors for functional gene analysis. Mol Ther. 16, 698-706.

Weise, J.M., and Günes, C. (2008)e. Differential regulation of human and mouse Telomerase Reverse Transcriptase (TERT) promoter activity during testis deve-lopment. Mol. Reprod. Dev. Aug 26. [Epub ahead of print]

Wiegers, K., Schwarck, D., Reimer, R., and Bohn, W. (2008). Activation of the glu-cocorticoid receptor releases unstimulated PBMCs from an early block in HIV-1 replication. Virology 375, 73-84.

Zafrakas, M., Tarlatzis, B.C., Streichert, T., Pournaropoulos, F., Wölfle, U., Smeets, S.J., Wittek, B., Grimbizis, G., Brakenhoff, R.H., Pantel, K., Bontis, J., and Günes, C. (2008). Genome-wide microarray gene expression, array-CGH analysis, and telomerase activity in advanced ovarian endometriosis: a high degree of diffe-rentiation rather than malignant potential. Int. J. Mol. Med. 21, 335-344.

Non Reviewed Publications, Reviews and Book Chapters

Kim, E., Giese, A., and Deppert, W. (2008)e. Wild-type p53 in Cancer Cells: When a Guardian Turns into a Blackguard. Biochem. Pharmacol. [Epub 2008 Sep 3].

Nevels, M., and Dobner, T. (2008). Erreger-induzierte Tumoren. In: Klinische Infektiologie. R. Marre, M. Trautmann & W. Zimmerli, eds. (München • Jena, Verlag Urban und Fischer), pp. 53-66.

Schindler M. (2008). Evolution des lentiviralen Nef Proteins. VDM-Verlag, ISBN 978-3-639-01822-6.

van Lunzen, J. (2008). Ritonavir-boosted tipranavir (TPV/r) in combination with new drug classes. Hospital Pharmacy Europe 39, 14-16.

Schindler, M., Hohenberg, H. (2008). Virologische Fährtensuche. Kombinierte Mikroskopietechniken zur Abbildung von Virusinfekti0onen. Bioforum 6, 2-6.

Stocking, C. and Kozak, C.A. (2008). Murine endogenous retrovirus. Cell. Life Mol. Sci. 65, 3383-3398.

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2007

Diplomarbeiten

Bohlmann, M.: „Identifizierung und Charaktisierung neuer aviärer Hepatitis B – Virusisolate“, Diplomarbeit, Fakultät für Biowissenschaften (Biologie), Universität Heidelberg (2007).

Gießelmann, K.: „Funktionelle Analyse des zellulären Proteins OTT/RBM15“, Diplomarbeit, Fakultät für Biowissenschaften (Biologie), Universität Heidelberg (2007).

Hildebrand, J.: „Regulation der LINE-1 Expression in humanen Tumorzelllinien durch Wildtyp und mutiertes p53.“ Fachbereich Biologie, Diplomarbeit, Universität Hamburg (2007).

Jahn, M.: „Beeinflussung der Expression eine Reportergens durch promo-terflankierende transposable bzw. repetitive Sequenzen in einem episoma-len Vektorsystem.“ Fachbereich Biologie, Diplomarbeit, Universität Münster (2007).

Knauer, T.: „Charakterisierung nichtkodierender Transkripte in humanen Glioblastoma Zellen.“ Fachbereich Humanbiologie, Diplomarbeit, Universität Erlangen (2007).

Peters, C.: „Untersuchungen zur Wechselwirkung der Ubiquitin-spezifischen Protease USP7 mit dem E1B-55K Protein humaner Adenoviren.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Biologie, Universität Hamburg (2008).

Pieper, D.: „Untersuchungen zur adaptiven zellulären Immunantwort am Beispiel der HIV/HCV-Coinfektion.” Naturwissenschaften, Diplomarbeit, Universität Hamburg (2007).

Saker, J.: „Regulation und Funktion der Telomerase und der Telomerfaktoren während der Differenzierung und Proliferation.“ Fachbereich Biologie, Diplomarbeit, Universität Hamburg (2007).

Schaub, T.: „Analyse der Funktion der PEST-Domänen und der GSK-3ß vermit-telten Phosphorylierung für die Stabilität des SPOC1-Proteins“, Diplomarbeit, Institut für Biowissenschaften (Mathematisch Naturwissenschaftlicher Fachbereich), Universität Rostock (2007).

Simon, N.: „Funktionelle Interaktionen von p53 mit Telomer- und Telomerase-Komponenten und Regulation der Telomerase-Aktivität durch p53.“ Fachbereich Humanbiologie, Diplomarbeit, Uni Marburg (2007).

Wimmer, P. „Analyse der transformierenden Eigenschaften des E1B-55K-Proteins von Adenovirus Typ 5“. Naturwissenschaftliche Fakultät III. Universität Regensburg (2007).

Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten, Bachelorarbeiten

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Dissertationen

Bruhn, S.: „NAADP, CD38 und Ca2+-Signalling: Komplexes Wechselspiel bei der Aktivierung von T-Lymphozyten.“ Fachbereich Chemie, Dissertation, Universität Hamburg (2007).

Fries, B.: „Einfluss des HuR-Liganden APRIL auf die CD83 Expression.“ Dissertation, Universität Erlangen-Nürnberg (2007).

Friese, O.: „Ein Mausmodell für die fehlende Erkennung methylierter DNA durch das Mdb1-Protein (Methyl-CpG-bindendes Protein 1).“ Fachbereich Chemie, Dissertation, Universität Hamburg (2007).

Iwanski, G.: „Untersuchungen zur Rolle des Transkripstionsfaktors C/EBPα bei der normalen und aberranten Hämatopoese mit Hilfe der RNA-Interferenz Methode.“ Dissertation, Fachbereich Medizin, Universität Hamburg (2007).

Kinkley, S.: „SPOC1: Characterization of a Novel Nuclear Protein with Implications for a Functional Role in Mitotic Chromosome Condensation.“ Fachbereich Biologie, Universität Hamburg (2007).

Kriegs, M.: „Untersuchung des Einflusses des Nicht-Strukturproteins 5A des Hepatitis C Virus auf Zellwachstum, Apoptose und Karzinogese in vitro und in vivo.“ Institut für Biologie, Humboldt-Universität zu Berlin (2007).

Lupberger, J.: „Cultivation of Hepatitis B Virus Producing Cell Line HepG2.2.15 on Microcarrier and Functional Characterization of the Hepatitis B Virus Polymerase.“ Institut für Biologie, Humboldt Universität zu Berlin (2007).

Mhamdi, M.: „Characterization of the envelope-mediated Steps in the life cycle of hepatitis B viruses (Intrazellulärer Transport und Morphogenese der Hepatitis B Viren).“ Fachbereich Biologie, Universität Hamburg (2007).

Prechtel, A.: „Untersuchungen zur posttranskriptionellen Prozessierung der Messenger RNA von CD83, dem Reifungsmarker Dendritischer Zellen.“ Dissertation, Naturwissenschaftliche Fakultät II (Biologie), Universität Erlangen-Nürnberg (2007).

M. Ritter: „A landmark-based method for the geometrical 3D calibration of scan-ning microscopes.” Fachbereich Informatik, TU-Berlin (2007).

Riu-Garcia, A.: „Functional Analysis of Hepatitis B Virus with Mutations in the Envelope Proteins.” Fachbereich Chemie, Dissertation, Uni Hamburg (2007).

Sander, M.-S.: „Quantifizierung der viralen DNA-Synthese im produktiven Infektionszyklus humaner Adenoviren durch Echtzeit-PCR.“ Medizinische Fakultät der Universität Regensburg (2007).

Habilitationen

Dr. Günes, C.: „Regulation und Funktion der Telomerase in normalen und Tumorzellen: Erkenntnisse aus in vitro und in vivo Modellen.“ Fachbereich Medizin, Habilitation, Universität Hamburg (2007).

Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten,

Bachelorarbeiten

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Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten, Bachelorarbeiten

2008

Bachelorarbeiten

Boukari, H.: „Zelluläre Determinanten der HBV Replikation.“ Bachelorarbeit, Fachbereich Biologie, Universität Hamburg (2008).

Fischer, T.: „Charakterisierung des Adenovirus Serotyp 5 H5pm4182.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Biologie, Universität Hamburg (2008).

Godau, J.: „Analysen zum transformierenden Potenzial des E1B-55K-Proteins von Adenovirus Serotyp 5.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Biologie, Universität Hamburg (2008).

Hofmann, S.: „Analyse der Tre-Rekombinase im HIV-1 LTR.“ Bachelorarbeit, Fachbereich Biologie/Chemie der Justus-Liebig-Universität Giessen (2008).

Homann, L.: „Untersuchungen zur Dynaminabhängigkeit des Viruseintritts von DHBV im primären Entenhepatozyten-System.“ Fachbereich Biologie, Universität Marburg (2008).

Sekutowicz, M.: „Untersuchungen der Auswirkungen zellulärer Proteine auf den adenoviralen Infektionsverlauf in humanen Hepatozyten mittels siRNA-Techniken.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Biologie, Universität Hamburg (2008).

Shaikh, Y.: „Methodische Untersuchungen zur Erhaltung der eGFP Fluoreszenz in biologischen Proben, während der Kryoprozessierung für die korrelative Mikroskopie.“ Life Science, HAW Hamburg (2008).

Masterarbeiten

Kluth, A.: „p53-abhängige Differenzierung embryonaler Stammzellen in einem Maustumorzellmodell.“ Studiengang Molekulare Biologie, Masterarbeit, Universität Gelsenkirchen (2008).

Diplomarbeiten

Ching, W.: „Funktionelle Analysen zur Phosphorylierung des E1B-55K-Proteins von Adenovirus Serotyp 5 durch Casein Kinase II.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Chemie, Universität Hamburg (2008).

Deing, V.: „Einfluss des Tumorsuppressorgens p53 auf die Qualität der Reparatur von UV-induzierten DNA Schäden.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Universität Hamburg (2008).

R. Getzieh: „Microbial Biofilms in Natural Environments and Medicine. Development and Establishment of a new Technique for Correlative Light and Electron Microscopy.“ Umwelt und Technik, Universität Lüneburg (2008).

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Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten,

Bachelorarbeiten

Keyser, B.: „Untersuchungen zur subzellulären Lokalisierung und zum intrazel-lulären Transport von ICP0 und ICP4 des Herpes Simplex-Virus 2 (HSV-2).“ Diplomarbeit, Fachbereich Biologie, Universität Hamburg (2008).

Kömm, N.: „Analyse der Funktionen von pRb2/p130 und pRb/p105 in der Induktion zellulärer Seneszenz mittels RNA-Interferenz.“ Diplomarbeit, Fachbereich Biologie, Universität Hamburg (2008).

Kopp, F.: „Charakterisierung der Interaktion des viralen E1B-55K-Proteins mit Securin und dessen Einfluss auf den produktiven Infektionszyklus von Adenovirus Serotyp 5.“ Mathematisch Naturwissenschaftliche Fakultät. Universität Rostock (2008).

Korf, K.: „Zelluläre Parameter der Simian Virus 40 (SV40) Amplifikation in Abhängigkeit des p53-Status der Wirtszelle.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Universität Hamburg (2008).

Leinenkugel, F.: „Funktionelle Analysen der E1B-48K- und der -49K-Isoform des Adenovirus Typ 5 E1B-55K-Proteins.“ Fakultät für Biologie. Albert-Ludwigs-Universität Freiburg (2008).

Malitzky, A.: „Herstellung und Überprüfung eines neuartigen adenoviralen Vektorsystems.“ Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Chemie, Universität Hamburg (2008).

Schmid, M.: „Analysen regulatorischer Bereiche im E1B-55K Protein von Adenovirus Serotyp 5 und ihre Rolle im produktiven Replikationszyklus.“ Naturwissenschaftliche Fakultät III, Universität Regensburg (2008).

Dissertationen

Ehlers, C.: „Einfluss des RNA bindenden Proteins AUF1 auf die CD83 Expression.“ Dissertation, Fakultät für Mathematik, Informatik und Naturwissenschaften, Department Chemie der Universität Hamburg (2008).

Fischer, M.: „Etablierung eines in vivo Modells in Mus musculus (Linneaus 1758) zur Untersuchung der onkongene Wirkung des Fusionsproteins TEL1/AML1.“ Dissertation, Fachbereich Biologie, Universität Hamburg (2008).

Hermannstädter, A.: „Die duale Rolle von p53 in der zellulären Immortalisierung und der SV40 LT-vermittelten Transformation.” Fachbereich Chemie, Dissertation, Universität Hamburg (2008).

Horn, F.: „Hemmung der HIV-1-Rev- und HTLV-I-Rex-Funktion mit Hilfe von Inhibitoren der Desoxyhypusin-Synthase.“ Dissertation, Medizinische Fakultät, Universität Erlangen-Nürnberg (2008).

Iwanski, K.: „Funktionelle Interaktionen von p53 mit Telomerasekomponenten und Regulation der Telomeraseaktivität durch p53.“ Fachbereich Medizin, Dissertation, Universität Hamburg (2008).

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Habilitationen, Dissertationen, Diplomarbeiten, Masterarbeiten, Bachelorarbeiten

Majlessi, A.; „Sequenzanalyse von Hepatitis B Virus in Patienten mit fibrosie-render cholestatischer Hepatitis.“ Fachbereich Medizin, Universität Hamburg (2008).

März, A.: „Mutp53 Interaktom in einem murinen Tumorzellmodell.” Fachbereich Chemie, Dissertation, Universität Hamburg (2008).

Sieber, T.: „Untersuchung des funktionellen Potenzials der Adenovirus Typ 5 E1BN-Proteine.“ Naturwissenschaftliche Fakultät III, Universität Regensburg (2008).

Tögel, L.: „Epigenetische Funktion des mutierten Tumorsuppressors p53 in Mammakarzinoma.“ Fachbereich Biologie, Dissertation, Universität Hamburg (2008).

Olotu, C.: „Analyse von virusspezifischen Resistenzfaktoren und phylogenetischer Entwicklung von Hepatitis B Viren unter Therapie mit Adefovir.“ Dissertation, Medizinische Fakultät, Universität Hamburg (2008).

Habilitationen

Dandri, M.: „Chronic HBV infection and hepatocarcinogenesis – Lessons from in vitro and in vivo models.” Fachbereich Biologie, Universität Hamburg (2008).

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Lehrtätigkeit

■ MSc Molekulare & Funktionelle Biologie■ Wahlpflichtmodul: Molekulare Virologie und Zellbiologie (9 CP)

■ BSc Biologie ■ Fallstudie/ABKII: Methoden der molekularen Virologie und Zellbiologie

■ MSc Molekulare & Funktionelle Biologie■ Ringvorlesung: MSc Molekulare & Funktionelle Biologie. Virologie

■ Vorlesung: Molekulare Virologie und Zellbiologie (Universität Hamburg)■ Vorlesung: RNAi: Gene Regulation by small RNAs, Aufbaustudiengang

Molekularbiologie (ZMNH Hamburg)

■ Seminar: Research Highlights in Molecular Virology and Immunology■ Seminar: Experimental Approaches in Virology

■ FB Medizin Hamburg: Imagingtechniken in der Medizin

■ Vorlesung: Entstehung und Evolution des AIDS-Erregers HIV-1■ AIDS-Pathogenese

■ Seminar: Research Highlights in Molecular Hepatology and Immunology■ Ko-Organisator der Seminarreihe der Forschungsinitiative

„Leberentzündungen: Infektion, Immunregulation und Konsequenzen“

■ Seminar: Research Highlights in Leukemia■ Seminar: Experimental Approaches in Hematology

■ Seminar: Advances in Molecular and Cellular Biology (H. Will, I. Dornreiter)

■ Advances in Virus and Tumor Research (H. Will, I Dornreiter, G. Rohaly)

■ Praktika: Practical Introduction in Experimental Research (H. Will, I. Dornreiter, G. Rohaly, H. Staege)

■ Courses in Experimental Virology (H. Will, I. Dornreiter, G. Rohaly)

■ Vorlesung: Hepatitis B Viruses (H. Will)■ Papovaviruses (I. Dornreiter, G. Rohaly)

Thomas Dobner

AdamGrundhoff

Joachim Hauber

Heinrich Hohenberg

Michael Schindler

Hüseyin Sirma

Carol Stocking

Hans Will

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■ Alexander von Humboldt Stiftung (W. Deppert, T. Dobner, J. Hauber)■ Behring-Röntgen-Stiftung (J. Hauber)■ BioPark Regensburg (J. Hauber)■ Bundesministerium für Bildung und Forschung (H. Hohenberg, H. Will)■ Bundesministerium für Gesundheit, Berlin (H. Hohenberg)■ Charité – Universitätsmedizin Berlin (H. Will)■ Deutsche Forschungsgemeinschaft (T. Dobner, J. Hauber, H. Hohenberg,

C. Stocking)■ Deutsche Gesellschaft für Virologie (H. Will)■ Deutsches Krebsforschungszentrum Heidelberg (H. Will)■ Deutsche Krebshilfe (W. Deppert, A. Grundhoff)■ Eberhard Karls Universität Tübingen, Medizinische Fakultät (H. Will)■ Fond der Chemischen Industrie (H. Will)■ Frankfurter Stiftung Krebskranker Kinder (W. Deppert)■ Hamburger Kommission für Fragen der Gentechnik (A. Grundhoff)■ Helmholtz-Zentrum für Infektionsforschung (J. Hauber)■ Johann Wolfgang Goethe-Universität Frankfurt am Main, Medizinische

Fakultät (H. Will)■ Justus-Liebig-Universität Gießen, Medizinische Fakultät (H. Will)■ Leibniz-Gemeinschaft (H. Will)■ Ludwig-Maximilians Universität München, Naturwissenschaftliche Fakultät

(T. Dobner, H.Will)■ Max-Planck-Gesellschaft (W. Deppert, H. Will)■ Medizinische Hochschule Hannover (C. Stocking)■ Nationales Kompetenznetz HepNet (H. Will)■ Peter Hans Hofschneider Stiftungsprofessur (W. Deppert)■ Robert Koch Institut, Berlin (J. Hauber, H. Will)■ Robert-Koch-Stiftung (J. Hauber)■ Ruhr-Universität Bochum (W. Deppert)■ Ruprecht-Karls-Universität Heidelberg, Medizinische Fakultät (H. Will)■ Studienstiftung des Deutschen Volkes (T. Dobner, H. Will)■ Technische Universität Berlin, Fachbereich Vermessungstechnik (H. Hohenberg)■ Tumorzentrum Heidelberg/Mannheim (W. Deppert)■ Universität des Saarlandes, Medizinische Fakultät (A .Grundhoff)■ Universität Düsseldorf, Medizinische Fakultät (W. Deppert, J. Hauber)■ Universität Frankfurt, Medizinische Fakultät (J. Hauber)■ Universität Göttingen, Zentrum für Molekular Biowissenschaften (H. Will)■ Universität Hamburg, Medizinische Fakultät (W. Deppert)■ Universität Hamburg, Fakultät für Mathematik, Informatik und

Naturwissenschaften (W. Deppert, J. Hauber, H. Will)■ Universität Heidelberg (H. Will)■ Universität Leipzig, Medizinische Fakultät (H. Will)■ Universität Mainz, Fachbereich Medizin (H. Will)■ Universität Regensburg, Medizinische Fakultät (T. Dobner)■ Universität Rostock (H. Will)■ Wilhelm Sander-Stiftung (W. Deppert, T. Dobner)

Gutachtertätigkeitfür nationale

Vereinigungen

172

Gutachtertätigkeit

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■ American Society of Hematology (C. Stocking)■ American Society of Microbiology (C. Stocking)■ Associazione Italiana per la Ricerca sul Cancro (AIRC) (T. Dobner)■ Cancer Research UK (W. Deppert, C. Stocking)■ Chinesisch-Deutsches Zentrum für Wissenschaftsförderung, Peking (H. Will)■ Consiglio Nazionale delle Richerche, Rom (H. Will)■ European Inst. of Oncology, Milan, Italy (H. Will)■ Förderung der wissenschaftlichen Forschung, (FWF) Austria. (T. Dobner) ■ Fudan University, Shanghai, China (H. Will)■ Genesis Oncology Trust, Neuseeland (W. Deppert)■ German-Israeli Foundation (W. Deppert)■ INSERM, Institut Pasteur, Paris, France (H. Will)■ Israel Science Foundation (T. Dobner)■ Italian Association for Cancer Research (AIRC), Italy (H. Will)■ L’Agence Nationale de la Recherche, Paris (H. Will)■ Leukemic Research Foundation, London (C. Stocking)■ Medizinische Universität Wien (J. Hauber)■ Monash-University, Australia (H. Will)■ Nanyang Technological University, Singapore (H. Will)■ National Health Research Institute, Hinschu, Taiwan (H. Will)■ National Research Foundation, Pretoria, South Africa (H. Will)■ Northern Cancer Research Foundation, Canada (H. Will)■ Rhode Island University Hospital, USA (H. Will) ■ The Kay Kendall Leukemia Fund, London (C. Stocking)■ University of Adelaide, Australia (H. Will)■ University of Bratislava, Czech Republic (H. Will)■ University of New Foundland, Canada (H.Will)■ University of the Witwatersrand, Johannesburg, South Africa (H. Will)■ University of York, United Kingdom (W. Deppert)■ US-Israel Binational Science Foundation (T. Dobner)■ Wuhan University, China (H. Will)

Gutachtertätigkeitfür internationale

Vereinigungen

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Gutachtertätigkeit

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■ Japanese-German Cancer Workshop in Kyoto, Japan 2007 (Organisation)

■ Molecular Life Sciences. Herbsttagung der Gesellschaft für Biochemie und Molekularbiologie, Hamburg 2007 (Koorganisation)

■ Tagung: Adenovirus: Basic research and application. Ulm 2007 (Koorganisation und Chair)

■ DNA Tumor Virus Meeting. Madison, WI, USA. 2008 (Koorganisation und Chair)

■ Molecular Life Sciences. Herbsttagung der Gesellschaft für Biochemie und Molekularbiologie, Hamburg 2007 (Koorganisation)

■ SFB Symposium on viral Hepatitis, Gießen, 2008.

■ 14th International Runx Meeting 2007; Singapore, 19.-22. August 2007 (Chairperson)

■ 36th Annual Scientific meeting of the ISEH Society for Hematology and Stem Cells; Hamburg, 28.-30. September 2007 (Koorganisation)

■ Molecular Life Sciences: Herbsttagung der GBM; Hamburg, 16.-19. September 2007 (Chairperson)

■ IACRLRD Meeting – From Molecular Pathogenesis to Targeted Therapy in Leukemia and Sold Tumors; Freiburg, 7.-11. September 2007 (Chairperson)

■ XVII. Wilsede Meeting “Modern Trends in Human Leukemia” 14.-18. Juni 2008 (Koorganisation)

■ Summer School “Mechansims of early differentiation; embryonegenesis, myogenesis, and lymphohematopoiesis”; Barsinghausen, 1.-5. September 2008 (Chairperson)

■ 15th International Runx Meeting 2008; Provincetown, MA, USA. 14.-17. September 2008 (Chairperson)

■ American Society of Hematology (ASH) Meeting, San Francisco, CA, USA. 5.-9. Dezember 2008 (Chairperson)

WolfgangDeppert

ThomasDobner

Joachim Hauber

Hüseyin Sirma

Carol Stocking

174

Organisation von Meetings und Chairman-Tätigkeiten

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Hans Will

175

Organisation von Meetings und Chairman-Tätigkeiten

■ 4th Workshop „Hot Topics in Hepatology“, Modena, Italien, 25.-26.01.2007 (Chairman)

■ 3nd Workshop “Function of SPOC1 in Cells and Cancer”, Universität München, 24.-26.3.2007 (Co-Organisator and Chairman)

■ 2nd Sino-German Symposium on Hepatology “Viral Hepatitis from Basic Research to Clinical Application”, Wuhan, China, 18.-19.05.2007 (Chairman)

■ Meeting on “Molecular Life Science 2007” of the German Society of Bio chemistry and Molecular Biology”, Hamburg, 16.-19.09.2007 (Co-Organisator)

■ New insights in HBV Diversity, Pathogenesis, Diagnosis and Treatment, Ghent, Belgien, 12.-14.12.2007 (Chairman)

■ 3rd Workshop “Function of SPOC1 in Cells and Cancer”, Dortmund, 08.02.2008 (Co-Organisator and Chairman)

■ International Symposium on “Occult Hepatitis B virus Infection: Biology and Clinical Impact”, Taormina, Italien, 07.-08.03.2008 (Chairman)

■ 4th Virgil Symposium, Lyon Frankreich, 26.-27.05.2008 (Co-Organisator)■ International Symposium on Hepatitis B Virus Diversity, University

Witwatersrand, Johannesburg, Südafrika, 27.07-03.08.2008 (Co-Organisator and Chairman)

■ International Symposium on Viral Hepatitis and Liver Diseases, 20.-24.3.2009 Washington, USA

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■ Biochemistry and Cell Biology (W. Deppert)■ Journal of Hepatitis (Editorial Board, H. Will)■ Oncology (W. Deppert)■ Virus Genes (Editorial Board, H. Will)

Mitherausgabe wissenschaftlicher Zeitschriften

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German-Israeli Foundation for Scientific Research

and Development (GIF)

Tokyo Metropolitan Organization for Medical Research

Fondazione Poieo

EU

EU

EU

Bundesministerium für Bildung

und Forschung

Bundesministeriumfür Bildung

und Forschung

Bundesministeriumfür Bildung

und Forschung

Bundesministeriumfür Bildung

und Forschung

Internationale Förderung

„Structural, biophysical and functional Wolfgang Deppert studies on mutant p53 and its interaction with DNA“ „Risk assessment of hematopoietic stem Carol Stocking cell (HSC) genes for leukemia induction, referred to „the Study“.

Europäische Förderung Onkologische Adenoviren (Virobori) Thomas Dobner

„Mutant p53 as target for improved Wolfgang Deppert cancer treatment“

„Manipulating tumor suppression: Wolfgang Deppert a key to improve cancer treatment“

„Molecular signatures as diagnostic Wolfgang Deppert and therapeutic targets for disseminated epithelial malignancies“

„Entwicklung und Umsetzung eines Verwertungs- Heinrich Hohenberg konzeptes zur Verbesserung der schutzrechtlichen Sicherung und Professionalisierung der Verwertung von Forschungsergebnissen am Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie (HPI)“

„Analyse der cccDNA von Hepatitis B-Viren: Hans Will Bildung, Stabilität, pathogebetische Bedeutung und Modulation durch antivirale Medikamente“Verbund projekt: Kompetenznetz Infektions- krankheiten (Hep-Net)

„GO-Bio 2: Entwicklung und Kommerzialisierung Joachim Hauber eines biotechnologischen Verfahrens zur Eradikation proviraler HIV-1 DNA aus Patientenzellen“

„Behandlung der chronischen myeloischen Leukämie Joachim Hauber und HIV-Infektion: Analyse der Wirkungsweise von elF-5A (TP1)“

Drittmittelprojekte2007-2008

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Verbund TOMCAT – Entwicklung und Verbesserung Heinrich Hohenberg von spezifischen magnetischen Nanopartikel zur Detektion maligner Tumoren, Teilvorhaben HPI

Zuwendung für die Einrichtung einer Kooperationstelle Heinrich Hohenberg im Leibnitz- Zentrum für Infektionsforschung (LZIF), für die Zeit vom 01.11.2007 bis 31.03.2010

Deutsche Krebshilfe e.V. Bonn

„Studien zur Funktion von zellulären und viralen Proteinen Hans Will in ND-10 Kerndomänen bei akuter promyelozytischer Leukämie und anderen maligen Erkrankungen“

„Funktionsanalyse von SPOC1: Ein neuer mit der Hans Will Entstehung und Progression von Ovarialkarzinom assoziierter Transkriptionsregulator und mögliches Therapieziel von Tumorerkrankungen“

„Co-Regulation invasionsassoziierter Gene humaner Wolfgang Deppert Gliome durch das zelluläre Proto-Onkogen Ets-1 und Tumorsuppressor p53“ „Beteiligung von Tumorstamm-/progenitorzellen Wolfgang Deppert am Aufbau ihrer zellulären Umgebung und der metastatischen Nische“

„Dissecting the mechanism of apoptosis-resistance in Wolfgang Deppert hematopoietic malignancies expressing wild-type p53“

„Induktion zellulärer Seneszenz in Glioblastomzellen Wolfgang Bohn des Menschen“ „Regulation und Funktion der Telomerase in Cagatay Günes Mammakarzinomen im WAP-T Mausmodell“ „Funktionelle Analyse der Rex Transaktivierung Joachim Hauber des Humanen T Zell Leukämievierus Typ I“ „Mouse models to study the collaboration between FLT3 Carol Stockingactivation and RUNX1 mutations in AML induction“

178

Bundesministeriumfür Bildung

und Forschung

Behörde für Wissenschaft

und Forschung

Drittmittelprojekte

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Drittmittelprojekte

„Mausmodelle für Untersuchungen zu FLT3-abhängigen Carol Stocking Signaltransferwegen und kooperierenden Onkogenen, die für die Aufrechterhaltung einer akuten Leukämie erforderlich sind“

„Dissecting the mechanism of apoptosis-resistance in Carol Stocking hematopoietic malignancies expressing wild-type p53“

Einfluss des Tumorsuppressorgens p53 auf die Regulation Irene Dornreiter- der Reparatur von DNA-Doppelstrangbrüchen und die Nicholas Kopplung der Reparatur an den Zellzyklus (Folgeantrag)

Deutsche Forschungsgemeinschaft

„Hepatitis B-Viren als molekulare Sonden für die Hüseyin Sirma systembiologische Analyse der hepatozellulären Differenzierung und Polarität“

„Molekulare Mechanismen der infektiösen Hüseyin Sirma Aufnahme und des zellulären Transports von Hepatitis B-Viren und deren Bedeutung für den viralen Wirts- und Lebertropismus“

„Regulation und Funktion des schadeninduzierten Irene Dornreiter- Polymerase alpha-Primase-wtp53 Komplexes im Nicholas ATM/ATR-vermittelten intra-S-Phasen Kontrollpunkt“

„Analysis of the metastatic cascade using mouse Wolfgang Deppert models for ductal mammary carcinoma“

„Modulation of Tumor Angiogenesis by CEACAM1 Wolfgang Deppert in a Mouse Model for Mammary Carcinogenesis (WAP-T Mice)“

„Analysis off tumor progression and metastasis in Wolfgang Deppert transgenic mouse models for SV40 induced mammary carcinogenesis“

„Funktionelle Analyse der HIV – Rev Transaktivierung“ Joachim Hauber

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„Inhibition des eukaryotischen Initiationsfaktors Joachim Hauber elF-A aus Plasmodien und humanen Zellen – Mechanismen der Apoptose-Induktion und Zielstruktur für einen gemeinsamen Therapieansatzzur Behandlung der Malaria und der HIV-Infektion“

„Aktivitätskontrolle und zelluläre Funktionen Heinz Lother von Transglutaminasen“

„Role of viral microRNAs in Kaposi`s Sarcoma- Adam Grundhoff associated Herpesvirus (KSHV) infection and KSHV-associated Disease“

„Molekulare Analyse der CD83 Expression in Jan Chemnitz dendritischen Zellen“

Förderung durch Vereine und Stiftungen

„Disseminated Tumor Cells as Targets for Inhibiting Wolfgang Deppert Metastasis Epithelial Tumours“

„Studien zur Tumorgenese und Metastasenbildung Hans Will beim Eierstockkrebs: Beitrag des SPOC1-Proteins“

„Virus factories: Zentrale Plattformen für Hüseyin Sirma Assemblierung, Knospung und Sekretion von Viren“

„Epigenetische Inaktivierung von p53 in Wolfgang Deppert Glioblastomen des Menschen“

Seminarmittel und wissenschaftliche Retreats Heinrich Hohenberg

Mittel für Heinrich Pette Lectures 2007 Heinrich Hohenberg

„Hemmung der Herpes Virus Reaktivierung durch Heinrich Hohenberg PML-Kerndomänen-vermittelten epigenetischen Hüseyin Sirma Repression und ihre feinmorphologische Analyse.“ „Funktionelle Analyse der Interaktion des sehr frühen Joachim Hauber Herpes Simplex-Virus Regulatorproteins ICP0 mit der zellulären E3-Ligase SIAH-1“

„Hit & Run Transformation“ Thomas Dobner

180

Drittmittelprojekte

Verein zur Förderungder Krebsforschung e.V.

Horst MüggenburgStiftung

Stiftung zur Bekämpfungneuroviraler Erkrankungen

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181

„Bedeutung der Interaktion des HIV-1 Vpr mit dem Michael Schindler Glukokortikoidrezeptor für die virale Replikation und die neuronale AIDS Pathogenese“

„Induktion genomischer Instabilität als Grundlage Thomas Dobner Virus-vermittelter Onkogenese“

„Analyse von mutiertem p53 als Progressionsfaktor Wolfgang Deppert in einem WAP-mutp53 transgenen, induzierbaren Mausmodell für das Mammakarzinom“ „Analyse der Telomeraseregulation während der Ent- Cagatay Günes stehung und Progression von Mammakarzinomen“ „Adenovirus LDL-Rezeptor“ Thomas Dobner

„Untersuchung der Funktionen des pathogenen Adam Grundhoff Potentials der microRNAs des Epstein-Barr-Virus“

„Synergitische Wirkung der Transkriptionsfaktoren Carol Stocking MEF2C und ICSBP bei der Entstehung der akuten myeloischen Leukämie (AML)“

„Die Bedeutung der MEF2C und MEF2D Transkriptions- Carol Stocking faktoren bei der Entstehung der akuten Leukämie“

„Mausmodelle für die Entstehung der durch die (12;21) Carol Stocking Translokation verursachten akuten Iymphoblastischen Leukämie (ALL)“

„Mausmodelle für die Entstehung der durch die (12;21) Carol Stocking Translokation verursachten akuten Iymphoblastischen Leukämie (ALL)“ (Folgeprojekt)“

W. Sander-Stiftung

Erich und GertrudRoggenbuck-Stiftung

Hamburger Stiftung zur Förderung der Krebsbekämpfung

Deutsche José Carreras Leukämie-Stiftung e.V.

Drittmittelprojekte

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Dade Behring Marburg GmbH

Biomedizinische Forschungsgesell-schaft mbH Wien

Axxima Pharmaceuticals

ASTRAZENECA UKLimited

FEI Company

Bal-Tec AG

Media Cybernetics, Inc.

Point Electronics GmbH

Nikon GmbH

Industriekooperationen

Es sollen HBs-Expressionsvektoren hergestellt werden, Hans Will mit denen die mutierten HBs-Antigene der gelieferten Hepatitis B Virus DNAs in eukaryontischen Zellen exprimiert werden sollen.

„Untersuchung der subzellulären Lokalisation sehr Joachim Hauber früher Genprodukte des humanen Herpesvirus Typ 1 und Typ 2 (Herpes-Simplex-Virus 1 und 2)“

Zusammenarbeit auf dem Gebiet potentieller HSV Joachim Hauber Impfantigene

„Charakterisierung von durch Axxima ausgewählten Joachim Hauber chemischen Wirkstoffen zur Erforschung von Möglich- keiten zur Hemmung von HIV-Infektionen und Auf- klärung ihres Einflusses auf das zelluläre System; Analyse der Apoptose ausgelöst von durch Axxima ausgewählten chemischen Wirkstoffen in HIV-infizierten Zellen“

„WHERAS, AstraZeneca wishes to engage in scientific Carol Stocking research related to the response of mutant c-Kit receptors to the tyrosine kinase inhibitor AZD2171“

„Ausarbeitung gemeinsamer Konzepte zur „Systemischen Heinrich Mikroskopie“ inklusive der Entwicklung und Modifikation Hohenberg von elektronenoptischen Abbildungssystemen“

„Konzeption, Konstruktion und Realisation einer neuen Heinrich kompakten Hochdruckgefrierenanlage“ Hohenberg

Adaption des “Blind Deconvolution” – Algorithmus für Heinrich konventionelle und Environmental-REM-Daten Hohenberg

„Konzeption und Realisierung neuer Chip-Detektoren Heinrich für die hochauflösende 3D Raster-Elektronenmikroskopie“. Hohenberg

„Optimierung der Nikon Biostation für ein breites Heinrich Spektrum neuer biologischer Applikationen.“ Hohenberg

Drittmittelprojekte

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2007

Joachim Hauber, Ilona Hauber

Joachim Hauber, Ilona Hauber

2008

Ilona Hauber, Heinrich Hohenberg,

Joachim Hauber

„Piperidone derivatives and uses thereof“ (Europäische Patentanmeldung), gemeinsam mit Universität Würzburg (U. Holzgrabe, J. Klöckner)

„Use of a tailored recombinase for the treatment of retroviral infections“ (Europäische Patentanmeldung), gemeinsam mit MPI und Technische Universität Dresden (F. Buchholz, I. Sarkar, F.A. Stewart)

„Topical formulation for preventing sexual transmission of viral infection” (Europäische Patentanmeldung)

Erfindungsmeldungen und Patente

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Finanzielle Förderung Endstand 2007

Einnahmen Personalkosten Sachkosten Investitionen GesamtwertInstitutionelle Förderung 8.182.886 4.420.941 2.544.645 1.850.227 8.815.813

Summe Drittmittel 2.268.589 1.407.989 644.484 0 2.052.473

Gesamtes HPI 10.451.476 5.828.929 3.189.129 1.850.227 10.868.285

davon:

Bereich Grundfinanzierung 7.722.886 1.100.569 462.601 0 1.563.170 Bereich Gebäude und Technik 460.000 247.301 929.220 1.197.854 2.374.375 Bereich Wissenschaft 0 3.073.071 1.152.824 652.373 4.878.267

Summe Inst. Förderung 8.182.886 4.420.941 2.544.645 1.850.227 8.815.813

Drittmitteleinnahmen

MiGeb DFG 567.833 430.406 132.865 0 563.271MiGeb Diverse 142.114 10.198 106.934 0 117.132MiGeb EU 148.101 221.372 87.452 0 308.825MiGeb International 6.577 0 6.555 0 6.555MiGeb Krebshilfe 644.868 484.161 181.306 0 665.468MiGeb Öffentliche 430.182 47.584 8.156 0 55.740MiGeb Stiftungen 328.915 214.267 105.123 0 319.389MiGeb Unternehmen 0 0 16.093 0 16.093

Summe Drittmittel 2.268.589 1.407.989 644.484 0 2.052.473

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Finanzielle Förderung Vorläufiger Endstand 2008

Einnahmen Personalkosten Sachkosten Investitionen GesamtwertInstitutionelle Förderung 10.201.746 4.653.040 2.567.549 2.265.760 9.486.350

Summe Drittmittel 2.486.191 1.606.583 791.309 39.865 2.437.757

Gesamtes HPI 12.687.936 6.259.623 3.358.859 2.305.625 11.924.106

davon:

Bereich Grundfinanzierung 7.820.475 1.085.682 503.522 0 1.589.204 Bereich Gebäude und Technik 1.889.250 262.474 936.777 1.601.510 2.800.761 Bereich Wissenschaft 492.021 3.304.884 1.127.250 664.250 5.096.384

Summe Inst. Förderung 10.201.746 4.653.040 2.567.549 2.265.760 9.486.350

Drittmitteleinnahmen

InternationaleFörderungen 51.820 26.014 17.174 43.188EuropäischeFörderungen 308.056 174.293 63.475 237.768Bundesförderungen 605.039 254.094 263.337 39.865 557.296Deutsche Forschungs-gemeinschaft SPP 82.700 63.152 29.598 92.750Deutsche Forschungs-gemeinschaft 493.794 375.798 125.574 501.372Deutsche Krebshilfe 495.350 457.953 142.388 600.342Josè Carreras Stiftung 87.063 22.385 109.448Wilhelm Sander-Stiftung 65.200 22.055 10.692 32.747Förderung durchweitere Stiftungen 210.850 110.424 59.901 170.326Industriekooperationen 480 19.451 8.126 27.577diverse Mittelgeber 172.902 16.284 48.659 64.943

Summe Drittmittel 2.486.191 1.606.583 791.309 39.865 2.437.757

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11.01.07 Michael Schwarz, Institut für Pharmakologie und Toxikologie der Universität Tübingen, „Genotype-phenotype relationships in normal liver and liver cancer“

18.01.07 Marc Sitbon, Institut de Genetique Moleculaire de Montpellier CNRS UMR 5535, Montpellier, Frankreich, „HTLV-1 tropism and envelope receptor“

25.01.07 Martin Eilers, Institute for Molecular Biology and Tumor Research (IMT), Universität Marburg, „Control of cell proliferation and growth by myc proteins“

01.02.07 Felix Wieland, Biochemie-Zentrum der Universität Heidelberg (BZH), „Formation of a transport vesicle: players and rules“

08.02.07 Thomas von Zglinicki, Cellular Gerontology, University of Newcastle, School of Clinical Medical Sciences, Henry Wellcome Laboratory for Biogerontology Research, England, „Telomeres and mitochondria: Strange bedfellows in senescence“

02.03.07 Markus Manz, Institute for Research in Biomedicine (IRB), Bellinzona, Schweiz, „Human immune system Rag2-/-γc-/- mice: New options to study human lymphotropic viruses in vivo“

18.04.07 Thomas Sternsdorf, The Salk Institute for Biological Studies, La Jolla, California, „Artificial oncogenes as tools to dissect leukomogenesis“

19.04.07 Umberto Galderisi, Dept. Experimental Medicine, Second University of Naples, Napoli, Italy, „Cell cycle regulation in the biology of mesenchy-mal stem cells“

10.05.07 Nicole Fischer, Institut für Med. Mikrobiologie, Virologie und Hygiene, UKE Hamburg, „Virus detection and discovery using DNA microar-rays“

30.05.07 Michael Schindler, Institut für Virologie, Ulm, „The role of Nef in primate lentiviral pathogenesis“

Xavier Dervillez, Laboratoire de Biologie et Thérapeutique des Patho-logies Immunitaires, Hopital Pitié, Paris, „New vaccine strategies against HIV/AIDS: Development of (i) peptide or (ii) VLP-based multi-epitope vaccine candidates, expressing a hybrid FeLV-A p15E/HIV-1 gp41 MPER retroviral envelope“

14.06.07 Thomas Hofmann, Deutsches Krebsforschungszentrum (DKFZ), Heidel-berg, „HIPK2: an emerging regulator of DNA damage signaling“

21.06.07 Michael Nevels, Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, „Epigenetic control of human cytomegalovirus infection“

28.06.07 Herbert Tschochner, Lehrstuhl Biochemie II, Universität Regensburg, „Aspects of ribosome biosynthesis in eukaryotes“

Seminare

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Seminare

05.07.07 Stefan Müller, Max-Planck-Institute of Biochemistry, Dept. Molecular Cell Biology, München, „The SUMO system in the control of cell cycle progression and cell division“

12.07.07 Christoph Englert, Leibniz-Institut für Altersforschung – Fritz-Lipmann-Institut, Jena, „The Wilms tumor suppressor Wt1 in uroge-nital development: Insights from mice and zebrafish“

19.07.07 Rolf Renne, University of Florida, Dept. of Molecular Genetics and Microbiology, Gainesville, FL, U.S.A., „Kaposi´s sarcoma-associated herpesvirus-encoded microRNAs and their potential role in patho-genesis“

14.09.07 Urs Greber, Universität Zürich, Zoologisches Institut, „Computational systems analyses of adenovirus trafficking“

20.09.07 Larry Gerace, Depts. of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, U.S.A., „The nuclear lamina and signaling during myogenic differentiation“

27.09.07 Stefan Heinrichs, Dept. of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, U.S.A., „Identification of Structural Aberrations in Cancer by SNP Array Analysis“

01.10.07 Helga Hofmann, Dept. of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, U.S.A., „Identification of Structural Aberrations in Cancer by SNP Array Analysis“

11.10.07 Wulf Schneider, Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, „Adenoviral inhibition of TNF-r1-induced apoptosis signaling“

29.11.07 Stefan Müller, Max-Planck-Institute of Biochemistry, Dept. Molecular Cell Biology, München, „Ten years of SUMO-ubiquitin´s cousin“

17.12.07 Marta Szaszak, Leibniz-Institut für Molekulare Pharmakologie (FMP), Campus Berlin-Buch, „Intracellular trafficking of the angiotensin AT 1-receptor“

24.01.08 Walter Birchmeier, Max-Delbrueck-Center for Molecular Medicine (MDC), Berlin, „Wnt/beta-catenin signals in development and cancer“

31.01.08 Jo Milner, YCR P53 Research Group, Department of Biology, York, England, „Reciprocal regulation between p53 and SIRT1“

07.02.08 Ivo P. Touw, Experimental Hematology, Erasmus University Medical Center, Rotterdam, Niederlande, „Granulocyte colony-stimulating factor and its receptor in normal myeloid cell development, leukemia and related blood cell disorders“

14.02.08 Andreas Meyerhans, Institut für Virologie, Universität des Saarlandes, Homburg/Saar, „A microscopic and macroscopic view on viral infec-tions“

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Seminare

15.02.08 Juana Diez, Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain, „Effect of P-body components in positive strand DNA virus replication“

15.02.08 Kevin V. Morris, Dept. Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA, „Small RNA mediated transcriptional regu-lation of gene expression in human cells“

21.02.08 Mukesh Kumar, Dept. Plant Molecular Biology, MFPL, Wien, Österreich, „Arabidopsis Heat Shock Factors: Understanding their Function“

06.03.08 Alex Matter, Novartis Institute for Tropical Diseases, Singapore, „New drugs for neglected diseases“

03.04.08 Jochen Dahm-Daphi, Institut für Strahlenbiologie und Experimentelle Radioonkologie, Universitätsklinikum Hamburg-Eppendorf, „Regula-tion of DNA Double-Strand Break Repair“

10.04.08 Andreas Möller, Peter MacCallum Cancer Centre, Melbourne, Australien, „Siah regulates hypoxia signalling in cancer and inflammation“

17.04.08 Tristam Parslow, Emory University, Dept. of Pathology and Laboratory Medicine, Atlanta, GA, U.S.A., „Packaging the genome of influenza virus“

15.05.08 Bernard P. Mahon, Institute of Immunology, National University of Ireland Maynooth, „Mesenchymal stem cells, overcoming immunologi-cal barriers to therapeutic application“

22.05.08 Roger D. Everett, MRC Virology Unit, Glasgow, England, „STAT-1 and IRF-3 dependent pathways are not essential for repression of ICP0-null mutant herpes simplex virus type 1 in human fibroblasts“

12.06.08 Alexander Strom, Chronic Disease Program, Ottawa Health Research Institute (OHRI) Ottawa, Ontario, Canada, „Diabetes meets mad cow disease: Pancreatic ß-cells and cellular prion protein“

26.06.08 Barbara Schmidt, Institut für Klinische und Molekulare Virologie, Uni-versität Erlangen-Nürnberg, „Plasmacytoid dendritic cell (PDC) innate immune defense against HIV“

01.07.08 Dean Jackson, Faculty of Life Science, Manchester, UK, „Using advan-ced imaging techniques to explore nuclear structure and genome stabi-lity in mammalian cells“

03.07.08 Albert Heim, Medizinische Hochschule Hannover, Abt. für Virologie, „Disseminated adenovirus infections of immunosuppressed patients: Molecular virology and pathophysiology“

17.07.08 Gunhild Sommer, Medical University of South Carolina, Biochemistry & Molecular Biology, Charleston, South Carolina, USA, “The fusion protein OTTMAL induces R-loop formation and genomic instability“

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24.07.08 Tilman Heise, Medical University of South Carolina, Department of Biochemistry & Molecular Biology, Charleston, South Carolina, USA, „Translation elongation of cyclin D1 requires an RNA chaperone“

25.07.08 Stuart Le Grice, RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute – Frederick, U.S.A., „Reverse transcriptase-associated ribonuclease H – from single molecules to crystallization“

04.08.08 Rajesh K. Gaur, Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, U.S.A., „Modulation of alternative splicing: a potential therapy for cancer and other diseases“

18.09.08 Harald Wodrich, Institut de Genetique de Montpellier, Montpellier-Cedex, Frankreich, „Protein VI hires an ESCRT service for adenovirus during entry“

08.10.08 Wolfgang Kemmner, Max-Delbrück-Center for Molecular Medicine, Charité, Campus Buch, „Microarray examination of a transgenic mouse model for the ductal carcinoma in situ (DCIS) of the mammary gland“

13.11.08 Jan Münch, Institut für Virologie, Universitätsklinikum Ulm, „Natural inhibitors and enhancers of HIV infection“

25.11.08 Michael Nevels, Universität Regensburg, Institut für Medizinische Mikrobiologie und Hygiene, Regensburg, „Innate, Intrinsic and Epigenetic Control of Human Cytomegalovirus Infection“

Renate König, Infectious and Inflammatory Disease Centre, Burnham Institute for Medical Research, La Jolla, CA, USA, „HIV-host inter-actions – discovering host factors by high-throughput systems-based analysis“

Gülsah Gabriel, University of Oxford, Sir William Dunn School of Pathology, Oxford – United Kingdom, „Adaptation of a highly patho-genic avian influenza virus to the mammalian host: virus-host interac-tions“

Luka Cicin-Sain, Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaveron, OR, USA, „Environmental contribu-tors to immune aging“

03.12.08 Christine Stahl-Hennig, Deutsches Primatenzentrum Göttingen – DPZ, „Animal Models for Lentivirus Infection“

04.12.08 Ludger Hengst, Division of Medical Biochemistry Biocenter, Innsbruck Medical University, „CDK-inhibitors – connecting signal transduction and cell cycle control“

11.12.08 Peter Hemmerich, Leibniz-Institut für Altersforschung-Fritz-Lipmann-Institut e.V., Jena, „Superresolution microscopy of PML nuclear body assembly“

Seminare

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Mitglieder des Wissenschaftlichen Beirats 2007-2008

Name Institution Amtszeit

Prof. Dr. Hans Wolf (Vorsitzender)

Institut für med. Mikrobiologie und Hygiene, Universität Regensburg, Deutschland

Seit 2003

Prof. Dr. Thomas F. Schulz (stellv. Vorsitzender)

Institut für Virologie, Medizinische Hochschule Hannover, Deutschland

Seit 2008

Prof. Dr. Ueli Aebi Biozentrum M.E. Müller-Institut Universität Basel/Schweiz

Seit 2003

Prof. Dr. J. Valerie Bosch

ATV FO200, Deutsches Krebsforschungszentrum Heidelberg/Deutschland

Seit 2003

Dr. Roger Everett MRC Virology Unit, University of Glasgow, Scotland UK

Seit 2008

Prof. Dr. Wolfram Gerlich

Institut für Medizinische Virologie, FB 20, Universitätsklinikum Giessen, Deutschland

20.03.2003 - 19.03.2007

Dr. Dean A. Jackson University of Manchester Dept. Of Biomoleculare Sciences/UK

20.03.2003 - 19.03.2007 Neubestellung 20.03.2007 - 19.03.2011

MD, PhD Tristram G. Parslow

Microbial Pathogenesis HIV Pathogenesis, Pathology and Laboratory Medicine, University Hospital Atlanta, USA

23.01.2008 - 22.01.2012

Prof. Dr. med. Axel Rethwilm

Universität Würzburg, Institut für Virologie und Immunologie, Deutschland

20.03.2003 - 19.03.2007

(fettgedruckte Namen markieren aktuelle Mitglieder des Wissenschaftlichen Beirats)

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Mitglieder des Kuratoriums 2007-2008

Name Institution Amtszeit

Wilfried Mohr (Vorsitzender)

Medac GmbH, Hamburg Seit 2003

Senatsdirektor Gerhard Schneider (Stellv. Vorsitzender)

Behörde für Wissenschaft und Forschung (BWF), Hamburg

2003 - 2007

Leitender Regierungs­direktor Dr. Rolf Greve (Stell. Vorsitzender)

Behörde für Wissenschaft und Forschung (BWF), Hamburg

2008 - 2011

Prof. Dr. Hans Wolf Institut für Medizinische Mikrobiologie und Hygiene, Regensburg

Seit 2003

Prof. Dr. Michael Kramer

Bundesministerium für Gesundheit (BMG), Bonn

2003 - 2007

PD Dr. Lars Schaade Bundesministerium für Gesundheit (BMG), Berlin

Seit 2007

Regierungsdirektor Lutz Klinghammer

BMG, Berlin 2006 - 2007

Ministerialrätin Maria Becker

BMG, Berlin Seit 2007

Dr. Ingeborg Kirchhoff

Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz (BSG), Hamburg

Seit 2006

Regierungsdirektor Frank Laubert

Universität Hamburg 2003 - 2008

Regierungsdirektor Dr. Harald Schlüter

Universität Hamburg Seit 2008

Prof. Dr. Nikolaus Müller­Lantsch

Universität des Saarlandes, Homburg

2005 - 2008*

Prof. Dr. Hans Peter Mühlbach

Institut für Botanik, Universität Hamburg

2003 - 2008*

(fettgedruckte Namen markieren aktuelle Mitglieder des Kuratoriums)

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Die Kaufmännische Abteilung hat in den vergangenen Jahren einen umfassenden Wandel der personellen und organisatorischen Struktur vollzogen.

Der eingeschlagene Weg der Ausgliederung administrativer Prozesse im Personal-bereich ist in 2008 weiter entwickelt worden. Danach wird Anfang 2009 das Vertragsmanagement komplett durch einen externen Dienstleister übernommen, wodurch eine weitere Stelle in dieser Arbeitsgruppe eingespart werden kann.

Nachdem die umfangreichen Neu- und Umbauprojekte zwischen 2006 und 2008 von den Mitarbeitern der Technik realisiert werden konnten, wird in den nächs-ten Jahren das Bestandsmanagement in den Vordergrund treten. Die natürliche Fluktuation aufgrund von Ruhestandsregelungen, wird für eine Neuorganisation dieses Bereichs genutzt werden können. Konkret wird hier demnach von den zwei frei werdenden Stellen nur eine neu besetzt. Zur Verstärkung des Technikerteams ist geplant, eine Fachkraft aus dem Bereich Heizungs- und Installationstechnik einzustellen.

Die Dimensionierung der Arbeitsgruppe Finanz- und Rechnungswesen mit vier qualifizierten Fachkräften entspricht den institutsspezifischen Arbeitsanforde-rungen. Dabei wurde das Team in 2007 und 2008 durch einen externen Wirt-schaftsprüfer und Steuerberater unterstützt, der für Beratungs- und Fachaufgabe flexibel eingesetzt werden kann. Sowohl die Prüfungen des Finanz- und Rech- nungswesens durch behördliche Einrichtungen als auch durch externe Wirtschafts- prüfer haben zu keinen Beanstandungen geführt.

Das in den letzten beiden Jahren gebildete Einkaufsteam hat sich in seiner perso- nellen Zusammensetzung bewährt und eine hohe Akzeptanz in den Forschungs- abteilungen erreicht. Eine Mitarbeiterin des Einkaufsteams wird zukünftig im Vertretungsfall die Personalbetreuung übernehmen.

Die neu geschaffene Stelle des System-Administrators wird in 2009 auch verstärkt für die Unterstützung der wissenschaftlichen EDV im HPI eingesetzt werden. Dadurch kann eine weitere Personalaufstockung in der wissenschaftlichen EDV durch hausinterne Ressourcen abgedeckt werden. Die Stelle eines Auszubilden- den soll zum Herbst 2009 besetzt werden. Insbesondere der Aufbau eines EDV-gestützten Personalcontrollingsystems zur Steuerung des Stellenplans und des Budgets haben die Planungssicherheit im kaufmännischen Bereich erheblich ver- bessert.

Die Mitarbeiterin des Eingangsbereichs und der Warenannahme wird auch nach ihrer Verrentung weiter für das HPI tätig sein, so dass keine weitere Stelle für diese Funktion zur Verfügung gestellt werden muss. Dadurch kann ab Mitte 2009 zusätzlich eine Tierpflegerin eingestellt werden.

Der Umbau der Kaufmännischen Abteilung in Richtung einer serviceorientierten Dienstleistungseinrichtung ist weitgehend abgeschlossen.

Kaufmännische AbteilungLeiter: Dr. Volker Uhl

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Personal der Kaufmännischen Abteilung und Allgemeine Dienste

Dr. Volker Uhl

Kira FeldhinkelDorothea Stille*

Meike AlischSylvia BräuHelena KuhnLilia Spanagel

Kristin GesslerJörg Schinkel

Ingo Jackstien Katja KraußNina LenkPetra Plarre

Heiko Juretzka

(*zeitweise beschäftigt)

Leitung

Sekretariat

Finanz- und Rechnungswesen

Personalabteilung

Einkauf

EDV

Kaufmännische Abteilung

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Norbert Zangenberg, Dr. rer. nat.

Arne Düsedau, Dipl.-Ing.

Angela Homfeld, Dr. rer. nat.

Nicole Nolting, Dr. rer. nat.

Wissenschaftliche Stabsstellen

EDV

Großgeräte

Presse- undÖffentlichkeitsarbeit

Verstandsreferenz und LCI-Koordination

Carl-Heinz Lohm

Ingo GregorzewskiMichael HabenichtWolfgang MöllerOlaf Nehls

Technische Dienste

Leitung

Mitarbeiter

194

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Hasso MündtAnke DorendorfBeate MikscheMatthias Timmermann

Tierhaltung

Radica KaralicSvetlana Dragicevic-SlankamenacIrmgard HayeMargarete HouillonRadmilla KielMaria-Cristina MorenoMonika MüllerSemsa Reus

Spülküchen

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• Bestrahlungsgerät LISA Günes, Cagatay

• Bibliothek Bohn, Wolfgang Stellv.: Hintz-Malchow, Martina

• Biolog. Sicherheit Grundhoff, Adam Stellv.: Hauber, Ilona

• Datenschutz Düsedau, Arne

• Entsorgung Dornreiter, Irene

• Gleichstellung Iwanski, Alicja Stellv.: Neumann, Ute

• Großgeräte Düsedau, Arne

• Hygiene Beauftr. S-3 Hauber, Joachim

• Isotopenkoordinator Tessmer, Uwe Stellv.: Wiegers, Klaus-Jochen

• Laserschutz Düsedau, Arne

• Ombudspersonen Stocking, Carol Stellv.: Hohenberg, Heinrich

• S 3-Labor Hauber, Ilona

• Seminar Bohn, Wolfgang

• Sicherheit Tryba, Martin Stellv.: Nehls, Olaf

• Spülküche Bergholz, Ursula

• Tierhaltung Stocking, Carol Stellv.: Müller, Ursula

• Tierschutz Bruns, Michael

Beauftragte 2007-2008

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Page 208: Activity Report 2007/08

Gemälde von Edith Pette, das neben dem Abbild ihres Mannes hängt. Beide Gemälde schmücken nun das Foyer des HPI im neuen Ersatz- und Erweiterungsbau II.

Page 209: Activity Report 2007/08

Impressum

Verantwortlich Prof. Dr. Thomas Dobner für den Inhalt Dr. Heinrich Hohenberg

Redaktion Dr. Angela Homfeld Dr. Nicole Nolting

Grafik & Layout AlsterWerk MedienService GmbH Hamburg

Druck Hartung Druck + Medien GmbH Hamburg

Titelbild

Neu gestaltete Fassade des Seuchenlaborgebäudes

Page 210: Activity Report 2007/08

Tätigkeitsbericht2007/2008

Täti

gkei

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07/2

008

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