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Recent Advances in Pathogenic
Human Viruses
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3 Recent Advances in Pathogenic Human Viruses. H. Smithand
Charles E. Samuel
7S
Inhibitors of Histone Deacetylases.
CORRELATIONBETWEENISOFORMSPECIFICITYANDREACTIVATIONOFHIVTYPE1(HIV-1)FROMLATENTLYINFECTEDCELLS.
KellyHuber,Genevie`veDoyon,JosephPlaks,ElizabethFyne,JohnW.Mellors,andNicolasSluis-Cremer
15 HostProteinKu70BindsandProtectsHIV-1
IntegrasefromProteasomalDegradationandIsRequiredforHIVReplication.YingfengZheng,
ZhujunAo, BinchenWang, KalleshDanappa Jayappa,andXiaojianYao
29S
Impaired Infectivity of Ritonavir-resistant HIV Is Rescued
byHeat Shock Protein 90AB1. Pheroze Joshi and Cheryl A.
Stoddart
41 A Chimeric HIV-1 Envelope Glycoprotein Trimer with anEmbedded
Granulocyte-Macrophage Colony-stimulatingFactor (GM-CSF) Domain
Induces Enhanced Antibody and TCell Responses. Thijs vanMontfort,
Mark Melchers, Gozde Isik,SergeyMenis, Po-Ssu Huang, Katie
Matthews, ElizabethMichael,Ben Berkhout, William R. Schief, John P.
Moore, and Rogier W. Sanders
53 Identification of Interactions in the E1E2 Heterodimer
ofHepatitis C Virus Important for Cell Entry. Guillemette
Maurin,Judith Fresquet, Ophelia Granio, Czeslaw
Wychowski,Francois-Loc Cosset, and Dimitri Lavillette
65S
Identification of Cis-Acting Elements in the
3-UntranslatedRegion of the Dengue Virus Type 2 RNA That
ModulateTranslation and Replication. Mark Manzano, Erin D.
Reichert,Stephanie Polo, Barry Falgout, Wojciech Kasprzak, Bruce A.
Shapiro,and Radhakrishnan Padmanabhan
79S
Structural Characterization of the Crimean-CongoHemorrhagic
Fever Virus Gn Tail Provides Insight into VirusAssembly. D.
Fernando Estrada and Roberto N. De Guzman
The Journal of Biological ChemistryTABLE OF CONTENTS
2011 COMPENDIA COLLECTION: Recent Advances in PathogenicHuman
Viruses
S Online version of this article contains supplemental
material.
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Recent Advances in Pathogenic Human Viruses*H. SmithFrom the
American Society for Biochemistry andMolecular Biology, Rockville,
Maryland 20852
Edited and curated by Charles E. Samuel1
From the Department of Molecular, Cellular, and Developmental
Biology and the Bimolecular Sciences and Engineering Program,
University of California,Santa Barbara, California 93106
Written histories of virology research frequently start withthe
year 1796, when English physician Edward Jenner used
vac-cinia-laden pus, collected from the cowpox lesions of a
milk-maid, to inoculate a young patient against smallpox.
Mostaccounts are quick to acknowledge that prophylactic
inocula-tion did not begin with Jenner but had in fact been
practicedcenturies beforehand, in China and other regions, and that
theuse of cowpox lesions had been used even in Jenners day
inEurope. However, the details of Jenners experiments were
bothspectacular and well told. For example, after treating an
8-year-old patient with the cowpox inoculum, Jenner challenged
thepatient by intentionally inoculating himwith smallpox. By
care-fully recording his work, Jenner resonated with the
Enlighten-ment ideals of the scientific method. Ever since,
Jennersrecords have provided a convenient milestone in the
develop-ment of modern antiviral therapies.Virologys historical
importance to basic molecular biology
and the disciplines crucial role in health care around the
globehave been very much on the minds of the organizers of
RecentAdvances in Pathogenic Human Viruses, a meeting sponsoredin
part by the American Society for Biochemistry and Molecu-lar
Biology, thismonth inGuangzhou, China. Attendance at themeeting by
virologists from around the world not only reflectsthe
unprecedented therapeutic opportunities to be developedfrom studies
of viruses but also attests to the worldwide risksposed by viral
pathogens in our global age. A single case of adeadly viral disease
appearing in the English countryside today,or in a small Chinese
village, or anywhere else in the world, hasramifications for global
health that were unimaginable in Jen-ners day. Virology today must
be considered along dimensionsthat cross previously recognized
borders, including thosedrawn between countries, scientific
disciplines, political andeconomic ideologies, and even animal
species.Organization of the meeting on Recent Advances in
Patho-
genic Human Viruses in Guangzhou also reflects a
deliberatesentiment that the benefits and risks of
virology-relatedresearch are met best through collaborations that
are also builtto global proportions. The American Society for
Biochemistryand Molecular Biology, founded through a shared sense
ofexcitement for scientific discovery more than a hundred yearsago,
is proud to support the collegial spirit that has
broughtinternational scientists together in Guangzhou. We are
partic-
ularly delighted to offer meeting delegates the following
com-pendium of recent research findings in virology, presented
byauthors from around the world within the pages of The Journalof
Biological Chemistry (JBC). These current research paperstouch on a
variety of topics related to viral disease in humansand promise to
provide avenues for better understanding andcombating viral
pathogens. Beyond their clinical implications,these papers also
represent the light that virology inevitablycasts on processes
essential to the wide spectrum of intereststhat define modern
biochemistry and molecular biology.
Its All Very Retro
Groundbreaking investigation into the molecular biology ofthe
retroviruses, culminating in the early 1970s, establishedthat RNA
could be reverse-transcribed into DNA. This find-ing, made in
laboratories focusing on the nucleic acid metabo-lism of avian
viruses, broke the central dogma of molecularbiology that had been
pronounced by Francis Crick in the1950s. Cricks declaration that
the expression of genetic infor-mation within all cellular
organisms flows in a single-directionsynthetic pathway, from DNA to
RNA to protein, reflected thebasic conceptual framework by which
biologists had begun tounlock mechanisms of gene regulation. This
framework hadbeen built in large part through the work of basic
virologists.Similarly, it was a framework that was recast through
the workof virologists such as David Baltimore and Howard
Temin.2The discovery of reverse transcriptase, which
revolutionized
molecular biology, originated from studies of basic
retroviralbiology. In the field of retrovirology, the discovery
broughtcredibility particularly to Howard Temins thesis for the
exist-ence of a proviral intermediate for Rous sarcoma: that is,
for theexistence of a DNA form of the viral genome that
becamespliced into the host genome as a normal phase of viral
infec-tion. Temins proposal thus echoedDulbeccos identification
ofthe proviral intermediate in the replication cycle of the
polyoma(DNA) virus.2 The elucidation of reverse transcriptase,
alongwith other elements of the HIV-1 molecular machinery thatdrive
the retroviral life cycle, has paved the way for
therapeuticapproaches that have alleviated greatly the worldwide
burdenof illness caused by HIV-1. In addition, whereas drugs
thatinhibit the HIV-1 reverse transcriptase, integrase, and
proteasehave become important in the clinic, host factors that
affect
* Translated into Chinese by Xing Guo, University of California,
San Diego.To cite articles in this collection, use the citation
information that appears in
the upper right-hand corner of the first page of the article.1
To whom correspondence should be addressed. Tel.: 805-893-3097;
Fax:
805-893-5780; E-mail: [email protected].
2 The Nobel Prize in Physiology or Medicine 1975 was awarded
jointly toDavid Baltimore, Renato Dulbecco, and Howard Martin Temin
for theirdiscoveries concerning the interaction between tumour
viruses and thegenetic material of the cell. See
http://nobelprize.org/nobel_prizes/med-icine/laureates/1975/.
Accessed June 14, 2011.
2011 by The American Society for Biochemistry and Molecular
Biology, Inc. Printed in the U.S.A.PROLOGUE This paper is available
online at www.jbc.org
3
-
retroviral infection have also entered center stage in the
effortto understand and combat HIV-1 infection. Four of the
papersin this compendium attest to the diversity of HIV-1
targets.Going Pro-ViralThe proviral stage of the HIV-1 life
cycle
poses a great challenge in treating infection because the
viralgenome, once integrated into host DNA, can remain
quiescentthroughout therapeutic regimens that otherwise clear
theblood of virus. Latent infection is thus a constant threat
despitethe success of combinatorial drug strategies. HIV-1 latency
isthe issue at hand in the JBC paper from Huber and colleagues(1),
who explore the therapeutic potential of
pharmacologicallymanipulating epigenetic regulation ofHIV-1
proviral elements.Specifically, the recruitment of histone
deacetylase (HDAC) tothe long terminal repeats (LTRs) of theHIV-1
genome has beenlinked experimentally to the induction of HIV-1
latency, lead-ing Huber et al. (1) to question whether specific
isoforms ofhistone deacetylase might function differentially to
maintainproviral DNA in the form of quiescent chromatin. Indeed, in
Tcells isolated from aviremic HIV-1-infected individuals
under-going combination antiretroviral therapy, HDAC inhibitorshave
been able to induce chromatin relaxation as well as theactivation
of viral genes. Huber et al. (1) use a number of knowninhibitors of
HDAC activity to characterize nine distinctHDAC isoforms in Jurkat
cells with regard to HDAC inhibitionkinetics, association of HDAC
isoforms with provirus-contain-ing chromatin, and the effectiveness
of distinct HDAC inhibi-tors in activating latent virus in vitro.
In this way, the authorsreport that the inhibition of HDAC3 is
essential for activatingthe provirus but that HDAC1, although
amenable to inhibition,is not a suitable target in terms of
therapeutic HDAC inhibitionas reflected by activity in Jurkat T
cells. The investigators thusestablish the importance of
isoform-specific targeting in anyattempt to add HDAC inhibitors to
combination antiretroviraltherapy. Moreover, as there are distinct
cellular reservoirs thatmay support latent virus, the authors offer
their pharmacolog-ical method of profiling HDAC activities as a
relatively directmeans of surveying HDAC isoform-specific
activities in othercell types.Binding and Hijacking: HIV-1
IntegraseAlthough inhibi-
tors of theHIV-1 integrase have reached clinical trials, with
oneinhibitor having been approved for prescription, the roles
ofcellular proteins in regulating the viral integrase are not
fullyunderstood. Researchers have thus not yet tapped the
thera-peutic potential of inhibiting viral replication by blocking
any ofthe dozens of interactions that occur between the viral
enzymeintegrase and cellular proteins. One such cellular protein
isKu70, which participates in nonhomologous end-joining DNArepair
(a process implicated in retroviral infection) and hasbeen
identified, in the form of a p70/p80 dimer, as an autoanti-gen in
systemic lupus erythematosus. The current JBC paperfrom Zheng et
al. (2) adds a new dimension to Ku70 function-ality in HIV-1
replication, capitalizing on the recent identifica-tion of Ku70 as
a deubiquitinating enzyme. The authors estab-lish that the C
terminus of the integrase binds to N-terminalsequenceswithinKu70
and that Ku70 promotes the deubiquiti-nation of the
integrase.Moreover, integrasemediates the incor-poration of Ku70
into progeny virus. The Ku70 that is thushijacked upon virion
assembly ultimately promotes viral repli-
cation in distinct ways, protecting the integrase from
degrada-tion and mediating the genesis of viral nucleic acid
intermedi-ates upon cell entry.Aiding and Abetting: Cell Proteins
That Enable HIV Drug
ResistanceThe integrase is not the only viral enzyme to col-lude
with specific cellular proteins in the service of HIV repli-cation.
As the JBC paper from Joshi and Stoddart (3) shows,mutant forms of
the HIV protease that fail to promote full mat-uration of the
capsid protein (CA) can nevertheless attain enzy-mic functionality
and restore viral replication by associatingwith HSP90AB1, a
cellular heat shock protein. This cell pro-tein-virus protein
collusion, moreover, depends on the activa-tion status of the
infected T cell: rescue of protease function byHSP90AB1 occurs only
in T cells that have been activated.These results have fascinating
ramifications for drug develop-ment, as pharmacological inhibition
ofHSP90AB1 prevents therescue of impaired virus, and most
significantly, the mutantproteases at the center of Joshi and
Stoddarts experiments (3)are typical ofHIV that has become
resistant to theHIV antipro-tease drug ritonavir (Norvir).
Admittedly, the actual mecha-nism of viral rescue (e.g. chaperone
activity) by HSP90AB1 isnot yet clear, but CA conformation and
interaction with otherhost factors have been implicated in the
postentry stage of HIVinfectivity. In any event, the authors
illustrate the consummatemolecular behavior of HIV in usurping
cellular functions.Engineering of Anti-HIV Vaccines That Carry
Cytokine
SignalsThe development of antiretroviral drugs that
targetintracellular viral enzyme activity has been particularly
crucialto clinical efforts as effective HIV vaccines have remained
elu-sive. Indeed, one of the hallmark challenges in combating
AIDShas come from attempts to marshal viral immunogenicity
byexploiting the envelope glycoprotein complex (Env). In
theirprovocative report, vanMontfort et al. (4) directly address
HIVimmunogenicity, hypothesizing that a vaccine component thatcould
carry a direct signal of immune activationthat is, a sortof
chemokinemight better alert host defense systems againstthe
invading virus. The authors hypothesize that if the
overallantigenic message can be amplified, the host might mount
abetter defense. To investigate, the authors have constructed
achimericmolecule that consists of the Envprotein (in
triplicate)along with the immunostimulatory domain of the
granulocyte-macrophage colony-stimulating factor (GM-CSF). Upon
injec-tion into mice, the chimeric construct enhances both
humoraland cellular responses, compared with injection of Env
alone.The improved immunogenicity appears to reflect cytokine
sig-naling, as the chimera retains GM-CSF activity in vitro.Whether
other cytokinesmay prove effective as immunostimu-latory components
when chimerically partnered with Envremains to be seen.
Flaviviridae: Hepatitis C Virus and Dengue Virus
The Flaviviridae family includes scores of viruses, several
ofwhich are important pathogens in humans. The genomes
ofFlaviviridae viruses consist of single-stranded RNA of
positivepolarity but, unlike the case of the retroviruses, engender
noDNA intermediate. Two of the JBC papers included in
thiscompendium concern the biology of Flaviviridae. The
firstdealswith envelope glycoproteins of the hepatitis C virus (of
the
PROLOGUE: Recent Advances in Pathogenic Human Viruses
4
-
Hepacivirus genus), which remains a pathogen of global
impor-tance. The second focuses on the RNA genome of the
denguevirus (a Flavivirus), which is endemic to tropical and
subtropi-cal regions, causing tens of millions of infections per
year.HepatitisCVirus: Cell Entry andEnvelopeProteins E1andE2
Since discovery of the hepatitis C virus, in 1989,
investigationsinto the two HCV envelope glycoproteins (E1 and E2)
havebeen hampered by technological difficulties associated
withcarrying the virus in culture and by the high degree of
sequencevariabilitymanifest in both E1 and E2. Each of the two
envelopeproteins contains a large glycosylated N-terminal
ectodomainand a C-terminal transmembrane anchor, and despite the
chal-lenges of assessing discrete infective stages of HCV in cell
cul-ture, specific roles for each protein in cell receptor binding
andcell fusion have been suggested (5). One of the characteristics
ofthe E1 and E2 proteins, which was quite remarkable whenreported
in JBC well over a decade ago, is that discrete muta-tions that are
confined to the transmembrane domain of eitherprotein can affect
heterodimerization and virus replication (6).In their current JBC
paper, Maurin et al. (5) exploit the nat-
urally occurring variability of E1 and E2 sequences to
elucidatethe structural basis of intra- and intersubunit
interactions thatenable E1E2 heterodimers to subserve cell entry in
the infectiv-ity cycle. In particular, the authors have identified
combina-tions of E1 and E2 variants that can be coexpressed to
producestable heterodimers that are nevertheless nonfunctional.
Byapplying site-directed mutagenesis to such inactive
het-erodimers, moreover, the authors have determined sequencesand
residues, from both envelope proteins, that function con-certedly
to culminate in viral infectivity. In this way, the
authorsestablish structure-function relationships that go
beyondmerely descriptive terms such as transmembrane sequence
orectodomain. Indeed, discrete interactions within the E1 sub-unit
of the heterodimer, as well as interprotomeric interactionsthat
emanate from discrete residues within the E1 transmem-brane
sequence, prove essential to entry of the virus into thecell.
Ultimately, the authors indicate, functionalmechanisms ofviral
replication and infection are effected through subtle facetsof the
E1E2 interaction related to cell entry.Dengue Virus: Structure and
Function of the Viral Genome
Manzano et al. (7) focus their attention on the structure of
thessRNA genome of the dengue virus. Typical of
Flaviviridaegenomes, the dengue virus RNA is of positive polarity
(()-RNA), meaning that it can serve directly as message for
thetranslation of viral proteins. The genome also serves as a
tem-plate for the RNA-dependent RNA polymerase activity of theviral
nonstructural protein 5 (NS5), whereby a negative RNAstrand is
synthesized to serve as the template for production ofviral progeny
genomes. In this way, replication of the viralgenome is intimately
tied to the translation of viral proteins,with both processes
regulated by means of structural elementsthat arise through
intramolecular associations within thessRNA genome. One important
element, or group of elements,is the 3-untranslated region of the
genome.Manzano et al. (7) have employed a high-performance com-
puter algorithm that duly considers the stabilities and
probabil-ities of RNA folding intermediates as related to the core
regionof the 3-untranslated region. In addition, the authors use
in
vitro assays and immunofluorescence to compare the muta-tional
effects of RNA elements upon viral replication and trans-lation.
The authors markedly refine previous speculations con-cerning many
secondary structures of the viral genome, andthey offer stringent
evidence for regulatory roles of at least fiveoligonucleotide
sequences that cooperate differentially,depending upon whether the
()-ssRNA genome is directingtranslation or RNA-dependent RNA
synthesis. The latter proc-ess, moreover, appears to utilize
certain genomic RNA ele-ments during ()-RNA synthesis but not
()-RNA synthesis,whereas the former process (i.e. translation) may
utilize ele-ments in a context-dependent manner, depending on
whethertranslation occurs by canonical (i.e. 5-cap-dependent
transla-tion) or noncanonical mechanisms. The implications for
con-trolling dengue viral infections and basic molecular biology
areunclear, but the virology of the system is compelling whenviewed
in terms of mechanistic intricacies that are both elegantand
complex.
The Gn Protein of the Crimean Congo Hemorrhagic FeverVirus
Twomembers of the Bunyaviridae provide focus for the JBCpaper
contributed by Estrada and De Guzman (8), who provideatomic
resolution, in solution, for the cytoplasmic portion ofenvelope
glycoprotein Gn of the Crimean Congo hemorrhagicfever (CCHF) virus
(of the genusNairovirus). By comparison totheir previous assessment
of the similar protein in a member oftheHantavirus genus (also in
JBC (9)), the authors offer insightsinto the role of the Gn protein
in CCHF virion assembly. The100-residue cytoplasmic tail (in the
respective proteins fromboth viruses) contains two zinc finger-like
CCHC sequences,and the investigators establish by NMR that the four
definingresidues of the twoCys-Cys-His-Cysmotifs participate in
func-tional zinc coordination. The two CCHC motifs,
moreover,interact to form a very stable compact structure, such
that thetwo zinc fingers appear, according to solution dynamics,
tobehave as a single entity. Results for the Gn protein,
moreover,indicate that the zinc finger functionality is distinctive
in fur-ther respects. First, the -fold that typifies the CCHC
motifis supplemented, to the N-terminal side of the second
suchsequence, by a third (3) helix, so that the structure
ultimatelyconsists of four -folds and three helices. The
distribution ofcharged residues in the Gn 43 structure,
furthermore, is highlysuggestive of a function, typically
associated with zinc fingers, ininteracting with RNA: multiple
basic residues define one face ofthe globular structure, ideal for
interactions with nucleic acid,whereas acidic residues tend to
cluster on the opposing hemi-sphere of the structure. Indeed, the
researchers confirm that theelectrophoretic mobility of CCHF viral
RNA is significantlyretarded through interactionswith the
cytoplasmic tail ofGn.Theauthors thereby offer amodel inwhich the
native integral Gn pro-tein is envisaged to orchestrate the
assembly of lipid envelope andnucleoprotein components into progeny
virions.
Conclusion
The seven papers presented in this compendium touch ona variety
of issues related to the basic biology of distinctviruses. Many of
the observations can be related to potential
PROLOGUE: Recent Advances in Pathogenic Human Viruses
5
-
clinical opportunities, whereas others shed light not only
onviral processes of metabolism and replication, but also
onfundamental cell functions. The Journal of Biological Chem-istry
is proud to be a part of the unfolding history of virology,and we
continue to welcome important research findingsfrom virologists
from around the world. We hope that par-ticipants of the Recent
Advances in Pathogenic HumanViruses will enjoy reading the stories
told in the papers ofthis compendium, assembled especially for the
internationalmeeting in China.REFERENCES1. Huber, K., Doyon, G.,
Plaks, J., Fyne, E., Mellors, J. W., and Sluis-Cremer,
N. (2011) J. Biol. Chem. 286, 22211222182. Zheng, Y., Ao, Z.,
Wang, B., Jayappa, K. D., and Yao, X. (2011) J. Biol.
Chem. 286, 17722177353. Joshi, P., and Stoddart, C. A. (2011) J.
Biol. Chem. 286, 24581245924. van Montfort, T., Melchers, M., Isik,
G., Menis, S., Huang, P.-S., Mat-
thews, K., Michael, E., Berkhout, B., Schief, W. R., Moore, J.
P., and Sand-ers, R. W. (2011) J. Biol. Chem. 286, 2225022261
5. Maurin, G., Fresquet, J., Granio, O., Wychowski, C., Cosset,
F.-L., andLavillette, D. (2011) J. Biol. Chem. 286, 2386523876
6. Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L.,
Cacan, R., Bar-berot, B., Le Maire, M., Penin, F., and Dubuisson,
J. (2000) J. Biol. Chem.275, 3142831437
7. Manzano, M., Reichert, E. D., Polo, S., Falgout, B.,
Kasprzak, W., Shapiro,B. A., and Padmanabhan, R. (2011) J. Biol.
Chem. 286, 2252122534
8. Estrada, D. F., and De Guzman, R. N. (2011) J. Biol. Chem.
286,2167821686
9. Estrada,D. F., Boudreaux,D.M., Zhong,D., St. Jeor, S. C.,
andDeGuzman,R. N. (2009) J. Biol. Chem. 284, 86548660
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Inhibitors of Histone DeacetylasesCORRELATION BETWEEN ISOFORM
SPECIFICITY AND REACTIVATIONOFHIV TYPE 1(HIV-1) FROM LATENTLY
INFECTED CELLSSReceived for publication,August 30, 2010, and in
revised form, April 29, 2011 Published, JBC Papers in Press, April
29, 2011, DOI 10.1074/jbc.M110.180224
Kelly Huber1, Genevie`ve Doyon1, Joseph Plaks, Elizabeth Fyne,
John W. Mellors, and Nicolas Sluis-Cremer2
From the Division of Infectious Diseases, Department of
Medicine, University of Pittsburgh School of Medicine,Pittsburgh,
Pennsylvania 15261
Deacetylation of histone proteins at the HIV type 1 (HIV-1)long
terminal repeat (LTR) by histone deactylases (HDACs) canpromote
transcriptional repression and virus latency. As such,HDAC
inhibitors (HDACI) could be used to deplete reservoirsof
persistent, quiescent HIV-1 proviral infection. However,
thedevelopment of HDACI to purge latent HIV-1 requires knowl-edge
of theHDAC isoforms contributing to viral latency and
thedevelopment of inhibitors specific to these isoforms. In
thisstudy, we identify the HDACs responsible for HIV-1 latency
inJurkat J89GFP cells using a chemical approach that
correlatesHDACI isoform specificity with their ability to
reactivate latentHIV-1 expression. We demonstrate that potent
inhibition orknockdown of HDAC1, an HDAC isoform reported to
driveHIV-1 into latency, was not sufficient to de-repress the
viralLTR. Instead, we found that inhibition ofHDAC3was necessaryto
activate latent HIV-1. Consistent with this finding, we iden-tified
HDAC3 at the HIV-1 LTR by chromatin immunoprecipi-tation.
Interestingly, we show that valproic acid is a weak inhib-itor of
HDAC3 (IC50 5.5 mM) relative to HDAC1 (IC50 170M). Because the
total therapeutic concentration of valproicacid ranges from275 to
700M in adults, these datamay explainwhy this inhibitor has no
effect on the decay of latent HIV res-ervoirs inpatients. Taken
together, our study suggests an impor-tant role for HDAC3 in HIV-1
latency and, importantly,describes a chemical approach that can
readily be used to iden-tify the HDAC isoforms that contribute to
HIV-1 latency inother cell types.
Combination antiretroviral therapy (cART)3 can effectivelyreduce
plasmaHIV-1 to undetectable levels. However, upon itsinterruption,
there is usually a rapid rebound of viremia (1).This viremia is
thought to arise from latently infected reservoirssuch as memory
CD4() T cells or CD34() multipotenthematopoietic progenitor cells
(25). Therefore, any long termtherapeutic strategy targeted toward
eliminating HIV-1 infec-
tion must include compounds that purge the latent viral
reser-voirs thereby rendering them susceptible to cART.HIV-1 can be
maintained in a latent state by multiple differ-
ent mechanisms that inhibit virus gene expression after
inte-gration into the cellular DNA (68). For example,
epigeneticmodifications at or near the HIV-1 5-long terminal
repeat(LTR) can induce chromatin condensation that diminishes
theaccessibility of the HIV-1 promoter to transcription factors.
Inthis regard, it has been well documented that different
tran-scription factors can recruit histone deacetylase
(HDAC)enzymes to the HIV-1 LTR where they promote
chromatincondensation by deacetylating the -amino groups of lysine
res-idues in histone tails (914). Eleven distinct
zinc-dependentHDAC isoforms have been identified in humans. These
can beclassified into four families, namely class I (HDAC13 and
-8),IIa (HDAC4, -5, -7, and -9), IIb (HDAC6 and -10), and
IV(HDAC11), which differ in structure, enzymatic function,
sub-cellular localization, and expression patterns (15). To
date,multiple studies have demonstrated that recruitment ofHDAC1 to
the HIV-1 LTR by different DNA-binding com-plexes is sufficient to
induce viral latency (914). However,HDAC2 and HDAC3 can also bind
to the HIV-1 LTR and mayalso play an important role in viral
latency (12, 16, 17).Treatment of latently infected HIV-1 cell
lines and/or
CD4() T cells from aviremic HIV-1-infected individuals
oncARTwithHDACI can lead to chromatin relaxation and induc-tion of
viral transcription (reviewed in Ref. 6). Therefore, HDA-CIs are
considered as potential therapeutic agents for purgingthe latent
viral reservoir in HIV-1-infected individuals. How-ever, the active
site structures of the HDAC family are largelyconserved, and many
HDACIs exhibit activity against multipleHDAC isoforms. For example,
suberoylanilide hydroxamic acid(SAHA, vorinostat), an activator of
latent HIV-1 expression(1820), is a nonselective HDACI that
inhibits both class I andclass II HDAC isoforms (21). Because HDACs
exert crucialroles in numerous biological processes, including cell
cycle, celldifferentiation, and survival (15), simultaneous
inhibition ofmultiple HDAC isoforms will likely reduce their
therapeuticwindow by promoting undesirable side effects and/or
toxicity.Accordingly, the development of HDACI for anHIV-1
curativestrategy requires knowledge of the HDAC isoforms
contribut-ing to viral latency and the development of inhibitors
targetingthese isoforms. In this study, we use a chemical approach
thatcorrelates the isoform specificities of HDACI with their
abili-ties to reactivate latent HIV-1 expression to identify the
HDACisoforms responsible for HIV-1 latency in Jurkat J89GFP
cells.
S The on-line version of this article (available at
http://www.jbc.org) containssupplemental Fig. 1 and Tables 1 and
2.
1 Both authors contributed equally to this work.2 To whom
correspondence should be addressed: S817 Scaife Hall, 3550 Ter-
race St., Pittsburgh, PA 15261. Tel.: 412-648-8457; Fax:
412-648-8521;E-mail: [email protected].
3 The abbreviations used are: cART, combination antiretroviral
therapy;HDAC, histone deacetylase; HDACI, HDAC inhibitor; SAHA,
suberoylanilidehydroxamic acid; EGFP, enhanced green fluorescent
protein; FW, forward;REV, reverse; HIV-1, HIV type 1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp.
2221122218, June 24, 2011 2011 by The American Society for
Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
7
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The results from this study suggest that potent inhibition
ofHDAC3 may be important for reactivation of latent HIV-1.
EXPERIMENTAL PROCEDURES
MaterialsThe HDACI
4,5:8,9-dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol
(depudecin),suberoyl bis-hydroxamic acid,
cyclo[(2S)-2-amino-8-oxo-decanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperi-dinecarbonyl]
(apicidin),
cyclo-(D-Pro-L-Ala-D-Ala-L-2-amino-8-oxo-9,10-epoxydecanoic acid)
(HC toxin),
(2E)-5-[3-(phenylsulfonylamino)phenyl]-pent-2-en-4-ynohydro-xamic
acid (oxamflatin),
6-(1,3-dioxo-1H,3H-benzo[de]iso-quinolin-2-yl)-hexanoic acid
hydroxyamide (scriptaid), sodiumbutyrate, sodium 4-phenylbutyrate,
SAHA, valproic acid,
and[(R)-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-di-methyl-7-oxo-2,4-heptadienamide]
(trichostatin A) were ob-tained from Enzo Life Sciences (Plymouth
Meeting, PA).
4-Di-methylamino-N-(6-hydroxyamino)-6-(oxohexyl]-benzamide(CAY10398)
and N-phenyl-N-(2-aminophenyl) hexamethyl-enediamide (CAY10433)
were obtained from Cayman Chemi-cal Co. (Ann Arbor, MI). Sodium
1-naphthoate was obtainedfrom TCI America (Portland, OR).
Droxinostat was pur-chased from Sigma. Wortmannin was obtained from
Sigma.The AKT inhibitor IV was obtained from EMD
Biosciences(Gibbstown, NJ). The phospho-AKT antibody and AKT
an-tibody were obtained from Cell Signaling Technology (Bos-ton).
The -actin antibody was obtained from Abcam (Cam-bridge,MA). DNA
oligonucleotide primers were synthesizedby Integrated DNA
Technologies (San Diego). The recombi-nant purified HDAC isoforms,
the Fluorogenic HDAC assaykit, and the HDAC assay substrates were
purchased fromBPS Bioscience (San Diego). The J89GFP cells were a
kindgift from Dr David Levy.HDAC Activity AssaysThe lysine
deacetylase activity of
HDAC19 was assessed using the fluorogenic HDAC assay(BPS
Bioscience) according to the manufacturers instructions.The HDAC3
used in this assay was complexed with humannuclear receptor
co-repressor 2 (NCOR2; amino acids 395489), which is an activating
co-factor of this HDAC isoform(31). All assays were carried out
under steady-state conditions,and the assay read-out was optimized
for linearity both as afunction of time and enzyme concentration.
Inhibition assayswere carried out in 384-well plates. The assay
volume was 25land contained 0.1 mg/ml BSA, 20 M substrate, and
varyingconcentrations of the HDACI. All HDACI were dissolved
inDMSO. The final concentration of DMSO did not exceed 5.0%(v/v).
The formation of the fluorescent product was measuredusing a
SpectraMax M2 plate reader (Molecular Devices). Theexcitation and
emission wavelengths were 360 and 450 nm,respectively. The
concentrations of HDACI required to inhibit50% of the deacetylase
activity of an HDAC isoform (i.e. IC50)were calculated by
regression analysis using SigmaPlot software(Systat Software, Inc.,
San Jose, CA).HDACI CytotoxicityJurkat cells were maintained in
RMPI
1640 medium supplemented with 10% FBS (Atlanta Biologi-cals),
0.3 mg/ml L-glutamine, 100 units/ml penicillin, and 100g/ml
streptomycin. HeLa and 293T cells were maintained inDMEMmedium
supplementedwith 10% FBS, 0.3mg/ml L-glu-
tamine, 100 units/ml penicillin, and 100 g/ml
streptomycin.CD8()-depleted peripheral bloodmononuclear cellswere
iso-lated from fresh whole blood (100 ml) of HIV-negative
individ-uals, as described previously (38). To determine HDACI
cyto-toxicity, 1 104 cells were plated in 96-well plates with
varyingconcentrations of drug. Following a 24-h incubation period,
cellviability was measured using either the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Roche
Applied Sci-ence) or CellTiter 96 proliferation (Promega, Madison,
WI)assay. The concentration ofHDACI that decreased cell viabilityby
50% (i.e. 50% cytotoxic concentration (CC50)) was calculatedby
regression analysis using SigmaPlot software.Reactivation of Latent
HIV-1 by HDACIJ89GFP cells are a
Jurkat T-cell line that contains a stably integrated,
full-lengthHIV-1 provirus (strain 89.6) with an enhanced green
fluores-cent protein (EFGP) reporter incorporated into the
viralgenome (22). The viral genome in these cells is
transcriptionallysilent. However, upon stimulation with tumor
necrosis factor or HDACI, viral transcription was activated, and
viral expres-sion can be measured by EGFP production. We chose this
celllinemodel ofHIV-1 latency because the absence of viral
expres-sion was not due to mutations in either the Tat-TAR axis
(e.g.the ACH2 cell line (34) and the U1 promonocytic cell line
(36))or in the 5-LTR (e.g. the JK cell line (35)). The J89GFP
cellswere maintained in RMPI 1640 medium supplemented with10% FBS,
0.3 mg/ml L-glutamine, 100 units/ml penicillin, and100 g/ml
streptomycin. 5 105 cells/ml cells were plated in6-well plateswith
varying concentrations ofHDACI for 672h.The PI3K and Akt inhibitors
wortmannin and Akt inhibitor IV(AI4) were used at concentrations of
100 nM and 10M, respec-tively. The cells were thenwashed in PBS,
fixed in 4%paraform-aldehyde, and stored at 4 Cuntil analysis.
Reactivation of latentHIV-1 was determined by quantifying the
percentage of EGFP-positive cells using a FACScan flow cytometer
with FACSDivasoftware (BD Biosciences).DNA Microarray Analyses5 105
J89GFP cells were
treated with 200 nM SAHA, oxamflatin, scriptaid, and apicidinfor
24 h. Control experiments included J89GFP cells grown inthe absence
of HDACI and Jurkat cells infected with HIV-1(multiplicity of
infection of 1) for 24 h. Total cellular RNA wasextracted from
these cells using the RNeasy Plus RNA extrac-tion kit (Qiagen Inc.)
according to themanufacturers protocol.RNA quantification, quality
assessment, and DNA microarrayanalyses were carried out by
PhalanxBio, Inc. (Palo Alto, CA),using the Human Whole Genome
OneArrayTM microarray.Each treatment condition and control were
assessed in dupli-cate biological replicates, and all samples were
run in duplicatetechnical replicates on the arrays. Data analysis
was performedby PhalanxBio, Inc., using Rosetta Resolver
software.siRNAKnockdownsiRNAs targetingHDAC1,HDAC2, and
HDAC3, as well as a control scrambled sequence controlsiRNA,
were purchased from Qiagen (SA Biosciences). TheJ89GFP cells were
transfected with 60 nM siRNA using theNeon Transfection System from
Invitrogen, according tothemanufacturers protocol. The efficiency
of gene knockdownwas assessed by determining mRNA levels (described
below)and by Western blot analyses of protein expression.
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
8
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Quantitative Analysis of Gene TranscriptsRNA wasextracted from
treated cells using RNeasy Plus RNA extractionkit (Qiagen Inc.,
Valencia, CA) according to themanufacturersprotocol. RNA was
quantified using a Nanodrop 2000, and200400 ng of total RNA was
used in each reaction. RNA wasamplified using the QuantiTect SYBR
Green RT-PCR kit(Qiagen Inc., Valencia, CA) and the DNA Engine
Opticonsystem (Bio-Rad). Initiated HIV-1 transcripts were
detectedusing primers TAR-FW (5-GTTAGACCAGATCTGAGCCT-3) and TAR-Rev
(5-GTGGGTTCCCTAGTTAGCCA-3).Elongated HIV-1 transcripts were
detected using primersTAT-FW (5-ACTCGACAGAGGAGAGCAAG-3) and TAT-REV
(5-GAGTCTGACTGTTCTGATGA-3). HDAC1 wasquantified using primers
HDAC1-FW (5-CCAGTATTC-GATGGCCTGTT-3) andHDAC1-REV
(5-TGTACAGCAC-CCTCTGGTGA-3). HDAC2 was quantified using
primersHDAC2-FW (5-ATAAAGCCACTGCCGAAGAA-3) andHDAC2-REV
(5-TCCTCCAGCCCAATTAACAG-3). HDAC3was quantified using primers
HDAC3-FW (5-TGGCTTCT-GCTATGTCAACG-3) and HDAC3-REV
(5-TCTCTGC-CCCGACTTCATAC-3). -Actin mRNA copies were used asthe
normalization control (10) and were quantified using theprimers
-actin-FW (5-GTCGACAACGGCTCCGGC-3)and -actin-REV
(5-GGTGTGGTGCCAGATTTTCT-3).Relative gene expression levels were
calculated using theC(T) method (23).Chromatin Immunoprecipitation
(ChIP) Assays2 106
J89GFP cells were fixed with 1% formaldehyde for 10 min atroom
temperature. ChIP assays were carried out using the EZ-ChIP assay
kit (Millipore Billerica, MA). Immunoprecipitationwas performed
using 5 g of antibody (Invitrogen) againstHDAC13 or -8. Rabbit
immunoglobulin G serum (5g, SantaCruz Biotechnology Inc., Santa
Cruz, CA) was used to controlfor nonspecific immunoprecipitation of
DNA. Forward(LTRB primer 5, 5-AGGTTTGACAGCCGCCTA-3) andreverse
(LTRB primer 3, 5-AGAGACCCAGTACAG-GCAAAA-3) primers specific for a
203-bp region in theHIV-1LTR that encompasses the NF-B-binding site
LTR were usedto detect a specific interaction between an HDAC
isoform andHIV-1 DNA, as described previously (10).
RESULTS ANDDISCUSSION
Potent Inhibition of HDAC1 Is Not Sufficient to ReactivateLatent
HIV-1Previous studies have demonstrated thatrecruitment of HDAC1 to
the HIV-1 LTR by different DNA-binding complexes is sufficient to
induce viral latency (911,13, 14). Accordingly, we hypothesized
that a potent inhibitor ofHDAC1 would reactivate HIV-1 expression
in the J89GFP cellline model of viral latency. Initially, we
screened 16 structurallydiverse HDACI (Fig. 1) for the following:
(i) their inhibitoryactivity against recombinant purified HDAC1;
(ii) their cyto-toxicity (CC50) in Jurkat cells; and (iii) their
ability to reactivateHIV-1 expression in J89GFP cells (Table 1).
For point iii, thehighest possible sub-cytotoxic concentration
ofHDACI (deter-mined from the cytotoxicity assessments) was used.
Of the 16HDACI tested, 6 (apicidin, HC toxin, scriptaid,
oxamflatin,SAHA, and trichostatinA) exhibited potent activity
(IC50100nM) against purified HDAC1. Of these, only three
(oxamflatin,
apicidin, and trichostatin A) were able to stimulate
HIV-1expression bymore than 5% in the J89GFP cells, asmeasured
byflow cytometry analysis of EGFP expression. In this regard,
itshould be noted that apicidin, scriptaid, oxamflatin, and
SAHAexhibited similar CC50 values and were tested in the
J89GFPcells at identical concentrations, therefore allowing for a
directcomparison of their ability to reactivate latent HIV-1
expres-sion. Interestingly, valproic acid and sodium butyrate
werefound to be relatively weak inhibitors of HDAC1 (IC50 175M),
but each elicited a different effect in the J89GFP cells asfollows:
1 mM valproic acid reactivated HIV-1 expression inonly 4.3% of
cells; 1 mM sodium butyrate reactivated HIV-1expression in 66.4% of
cells. Taken together, these HDACIscreening studies show that
inhibition of HDAC1 is not suffi-cient to reactivate HIV-1
expression in J89GFP cells.Inhibition of HDAC3 Correlates with the
Reactivation of
Latent HIV-1Based on the data described above, we next car-ried
out in-depth analyses on theHDACIs apicidin, oxamflatin,scriptaid,
and SAHA (Fig. 2). Each of these HDACIs exhibitsimilar potency
against purified HDAC1 and similar cytotoxic-ity (CC50) values in
Jurkat cells (Table 1). However, at concen-trations of inhibitor
ranging from 0 to 500 nM, apicidin andoxamflatin reactivated HIV-1
expression in the J89GFP cells ina dose-dependent manner, whereas
SAHA and scriptaid elic-ited no effect (Fig. 2A). A time course
experiment demonstratedthat this lack of activity was not due to an
early or late EGFPpeak that was missed at the 24-h time point used
in the dose-response experiments (Fig. 2B). Because the EGFP
expressionquantitated in Fig. 2,A and B, only provides information
on thetranslated protein, we also used quantitative RT-PCR to
assessthe formation of HIV-1 RNA transcripts (Fig. 2C).
Consistentwith the flow cytometry analyses, oxamflatin and apicidin
sig-nificantly increased the abundance of elongated HIV-1
tran-scripts, but not initiated transcripts, compared with
untreatedJ89GFP cells. By contrast, scriptaid and SAHA did not
signifi-cantly increase the formation of either initiated or
elongatedHIV-1 transcripts.We also carried out cDNAmicroarray
analyses to determine
themagnitude and the extent of global gene expression
changesobserved in the J89GFP cells after 24 h of treatmentwith 200
nMoxamflatin, scriptaid, SAHA, or apicidin. Control
experimentsincluded untreated J89GFP cells and Jurkat cells
infected withHIV-1 for 24 h. The cDNA microarray data are provided
insupplemental Tables 1 and 2 and Fig. 1. SAHA and scriptaidwere
found to significantly (p 0.01) up- or down-regulate 3and 1% of all
genes compared with untreated cells, respectively.One study
reported that SAHA altered regulation in at least22% of genes in
CEM cells; however, a much higher concentra-tion of drug (2.5M) was
used which caused50% of cell deathafter 24 h (30).Oxamflatin and
apicidin resulted in gene expres-sion changes in 11 and 8% of all
genes compared withuntreated J89GFP, respectively. These higher
gene expressionlevels compared with scriptaid and SAHA could be due
to inhibi-tionofdifferentHDACisoforms(seebelow)and/or
theexpressionof HIV-1 proteins in the J89GFP cells. Microarray DNA
analysesrevealed that 2.7% of all genes displayed altered
expressionchanges in HIV-1-infected Jurkat cells compared with
uninfectedcells. Nevertheless, these data indicate that SAHA and
scriptaid
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
9
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were taken up into the J89GFP cells and induced gene
expressionchanges. However, at the concentrations tested, they
lacked theability to induce expression of latent HIV-1.
To gain insight into the mechanisms by which oxamflatinand
apicidin, but not scriptaid and SAHA, reactivated latentHIV-1, we
determined the in vitro inhibitory activity of each of
FIGURE 1. Chemical structures of HDACI used in this study.
TABLE 1In vitro activity against HDAC1, cytotoxicity in Jurkat
cells, and reactivation of latent HIV-1 in J89GFP cells by
structurally diverse HDACI
HDACI IC50 against HDAC1 Cytotoxicity CC50Reactivation
dosea% inhibition of HDAC1 at
reactivation doseb% EGFP-positive J89GFP
cells (after 24 h)M M M
HC toxin 0.000154 0.05 0.005 99.7 1.3Apicidin 0.000299 10.0 0.5
99.7 40.6Oxamflatin 0.003959 6.0 0.5 99.2 31.2Scriptaid 0.006421
6.0 0.5 98.7 3.2SAHA 0.0137 10.0 0.5 97.3 2.5TSA 0.0169 0.10 0.05
74.7 17.2M344 0.0941 0.5 0.1 51.5 3.1CAY10398 1.7780 1.0 0.5 21.9
0.9MC1293 4.245 10.0 5 54.1 2.7CAY10433 9.36 1000 10 51.6 5.2SBHA
4.54 100 100 95.6 57.5Depudecin 25.33 5.0 1 3.8 1.3Sodium
1-naphthoate 200.6 10 10 4.75 1Valproic acid 171 10,000 1000 85.4
4.3Sodium butyrate 175 10,000 1000 85.1 66.4Sodium 4-phenylbutyrate
162 10,000 1000 86.1 2.5
a The highest nontoxic concentration of HDACI (determined from
the cytotoxicity assays in Jurkat cells) was administered to the
J89GFP cells to determine the inhibitorsability to reactivate
latent HIV-1.
b Maximum possible inhibition of HDAC1 at the concentration of
inhibitor used in the reactivation experiments in J89GFP cells is
shown (the actual inhibition in theJ89GFP cells is likely to be
significantly less due to inefficient cellular uptake and
nonspecific protein binding).
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
10
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the HDACI against recombinant purified HDAC1 and -2, the3-NCOR2
complex, and -49 (Table 2). In general, each of theHDACI exhibited
little or no activity against the class II HDAC
isoforms, although scriptaid exhibited excellent activity
againstHDAC6 (IC50 34 nM) and oxamflatin activity againstHDAC6(IC50
390 nM) and HDAC7 (IC50 840 nM). By contrast, allfour of the HDACI
were found to be very potent inhibitors ofthe class I HDAC isoforms
1, 2, and 8 (IC5020 nM). Interest-ingly, oxamflatin and apicidin,
both of which induced HIV-1outgrowth in the J89GFP cells, also
potently inhibited theHDAC3-NCOR2complex (IC5010nM). By contrast,
scriptaid(IC50 320 nM) and SAHA (IC50 600 nM) were 100-foldless
active against the HDAC3-NCOR2 complex. Based on ourIC50
calculations, a 200 nM dose of apicidin and oxamflatinwould inhibit
95% of the deacetylase activity of HDAC13and -8. (In the J89GFP
cells, these values would likely be signif-icantly less due to
inefficient inhibitor uptake and nonspecificprotein binding.) By
contrast, a 200 nM dose of scriptaid andSAHA would inhibit 95% of
HDAC1, -2, and -8 but wouldonly inhibit 25 and 18% of the
deacetylase activity of HDAC3,respectively. These values for
HDAC3would increase to 45 and36%, respectively, at a dose of 500
nM. Taken together, thesedata provide strong evidence that potent
inhibition of HDAC3is required to reactivate the expression of
HIV-1 in the J89GFPcells. The data also suggested that increasing
the concentra-tions of scriptaid and SAHA to allow inhibition of
HDAC3would result in the activation of latent HIV-1 in the
J89GFPcells. Indeed, in Fig. 2Dwe show that 2 M scriptaid and
SAHApromote activation of latent HIV-1 in the J89GFP. At this
con-centration, the total inhibition of HDAC3 approaches 76 and70%
for scriptaid and SAHA, respectively. To further assess
theimportance of HDAC3 inhibition in the reactivation of
latentHIV-1 infection, we assessed the ability of droxinostat to
reac-tivate latent HIV-1 expression in the J89GFP cells.
Previousstudies reported that this HDACI selectively inhibited
HDAC3and -8 but notHDAC1 and -2 (37). Indeed, we found that
droxi-nostat is a reasonably potent inhibitor of
recombinantHDAC3-NCOR2 complex and HDAC8 (IC50 2.0 and 3.0 M,
respec-tively) but shows only weak activity against HDAC1 and
-2(IC50 63 and 250 M, respectively) (Table 2). Of note,
droxi-nostat was found to reactivate latent HIV-1 expression in
adose-dependent manner (Fig. 3). At a 40 M concentration
ofdroxinostat, HDAC3 and -8 would be inhibited by 95%,whereas
therewould be only partial inhibition ofHDAC1 (38%)andHDAC2 (14%).
Taken together, these studies provide addi-
FIGURE 2. Reactivation of latent HIV-1 in J89GFP cells by
oxamflatin, api-cidin, scriptaid, and SAHA. A, J89GFP cells were
treated with varying con-centrationsofHDACI (0500nM), and
thepercentageof cells expressingEGFPwasquantitated after 24hbyFACs.
Error bars represent S.E. fromat least threeindependent
experiments. B, J89GFP cells were treated with 200 nM HDACI,and
thepercentageof cells expressingEGFPwasquantitatedatdifferent
timeintervals (075 h) by FACs. C, increase in HIV-1 RNA transcripts
in cells treatedwith 200 nM HDACI for 24 h as measured by
quantitative RT-PCR. Error barsrepresent S.E. fromat least three
independent experiments. The fold increasein transcript relative to
untreated cells is indicated above each bar for allfour HDACI. D,
J89GFP cells were treated with varying concentrations ofHDACI (0 2
M), and the percentage of cells expressing EGFP was quan-titated
after 24 h by FACs. Error bars represent S.E. from at least
threeindependent experiments.
TABLE 2In vitro activity of HDACI against class I and class II
HDAC isoforms
HDAC isoformIC50 against HDAC isoforms (nM )
Apicidin Oxamflatin Scriptaid SAHA Droxinostat Valproic
acidClass IHDAC1 0.30 0.15a1 3.96 0.87 0.64 0.09 13.7 0.15 63,000
171,000HDAC2 1.2 0.80 0.16 0.11 1.4 0.74 62.0 0.15 250,000
634,000HDAC3 0.98 0.22 10.3 1.2 607 93 869 0.15 2000 5,500,000HDAC8
0.26 0.09 0.37 0.15 14.5 1.1 6.8 0.15 5000 756,000
Class IIHDAC4 50,000 3800 1100 14,000 1500 50,000 NDb NDHDAC5
50,000 50,000 50,000 50,000 ND NDHDAC6 50,000 390 73 34 9 5500 760
ND NDHDAC7 50,000 840 39 2200 350 50,000 ND NDHDAC9 50,000 50,000
50,000 50,000 ND ND
a Data represent the mean S.D. from three replicate
experiments.b ND, not determined.
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
11
-
tional evidence that HDACI with specificity toward HDAC3can
reactivate latent HIV-1 expression in J89GFP cells.Several studies
have recently demonstrated that oxamflatin,
apicidin, scriptaid, and SAHA can reactivate latent HIV-1
indifferent cell lines and/or in resting CD4() T cells from
avire-mic patients (18, 19, 20, 32). In each of these studies
relativelyhigh concentrations (500 nM) of inhibitor were used. Of
note,each of these HDACI display significant toxicity in cell
lines(CC50 values range from 5 to 10 M) and in
CD8()-depletedperipheral blood mononuclear cells (CC50 values range
from0.1 to 7.5 M) (Table 3). In this regard, the small
therapeuticwindow of these HDACI highlights one potential
limitation fortheir inclusion in therapeutic combinations targeted
towardthe eradication of HIV-1.HDAC3 Resides at the HIV-1 LTR in
J89GFP CellsThe data
described above provide strong evidence that inhibition ofHDAC3
is important for the activation of latent HIV-1. How-ever, only two
studies have identified this HDAC isoform at theHIV-1 LTR (16, 17).
Accordingly, we performed ChIP assays inthe J89GFP cells using
antibodies specific for the class I HDACisoforms (Fig. 4A).We
detected a strong signal for HDAC1 and-3 indicating that both of
these HDAC isoforms resided at theHIV-1 LTR. We also detected a
weak signal for HDAC2 sug-gesting that this isoformmay also be
present at the HIV-1 LTR
in J89GFP cells. HDAC8 did not associate with the HIV-1
LTR.These findings are consistent with a recent study byKeedy et
al.(17), who also reported that HDAC13 resided at the HIV-1LTR in
J89GFP cells. Of note, this study also reported thatHDAC8 is
primarily sequestered in the cytoplasm and not thenucleus in J89GFP
cells (17). Quantitative real time PCR exper-iments confirmed the
presence of HDAC1 and -3, but notHDAC2, at the HIV-1 LTR (Fig. 4B).
Importantly, the occu-pancy of HDAC1 and -3 at theHIV-1 LTR in the
J89GFP cells islost upon treatment with apicidin (Fig. 4B) or
oxamflatin (datanot shown). To further assess the role of HDAC13 in
main-taining HIV-1 latency, we knocked down their gene
expression
FIGURE 3. Reactivation of latent HIV-1 in J89GFP cells by
theHDAC3-spe-cific inhibitor droxinostat. The chemical structure of
droxinostat is shown.The number of J89GFP cells expressing EGFP was
quantitated after 24 h byFACs. Error bars represent S.E. from two
independent experiments.
TABLE 3Cytotoxicity of HDACI in different cells
HDACI
CC50 (M)
Jurkata HeLaa 293TaCD8()-depleted
PBMCb
Apicidin 10.0 0.1 11.3 1.7 12.2 1.4 0.1Oxamflatin 5.9 0.1 9.4
0.8 6.0 1.0 0.3SAHA 10.0 0.1 11.3 0.3 14.1 0.2 7.5Scriptaid 6.1 0.3
8.9 1.8 5.8 0.2 0.7
a Data represent the mean S.D. from three replicate
experiments.b Data represent the mean from two independent
replicate experiments.
FIGURE 4. Role of HDAC13 in maintaining HIV-1 latency. A, ChIP
assaysidentify HDAC13, but not HDAC8, at the HIV-1 LTR in J89GFP
cells. Two dif-ferent HDAC1 antibodies (a and b) were used in the
ChIP assays. B, quantita-tive real timePCRexperiments
showenrichment (versus rabbit IgG) of HDAC1and -3 at the HIV-1 LTR
in J89GFP cells that is lost upon treatment with apici-din. PCR
data were normalized by quantification of the GAPDH promoter inthe
input samples. C, mRNA expression of HDAC13 after knockdown
bysiRNA. HDAC1* and HDAC2* reports on the mRNA levels in
experiments inwhichbothHDAC1and -2were knockeddownsimultaneously.
An siRNAwitha scrambled sequencewas used as a control. D,
reactivation of latent HIV-1 inJ89GFPcells after knockdownofHDAC1or
-2orHDAC1and -2. ThenumberofJ89GFP cells expressing EGFP was
quantitated after 24 h by FACs. Error barsrepresent S.E. from three
independent experiments.
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
12
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by siRNA (Fig. 4C). The magnitude of the siRNA-mediatedgene
silencing was confirmed by quantitative PCR analyses ofmRNA levels
(Fig. 4C) and by Western blot analysis (data notshown). Knockdown
ofHDAC3 resulted in significant and sub-stantial cell death that
prevented subsequent analyses of HIV-1latency. Interestingly, the
knockdown of either HDAC1 or -2,or a combination of HDAC1 and -2,
did not result in the reac-tivation of latentHIV-1 expression in
the J89GFP cells (Fig. 4D).These data provide strong supporting
evidence that inhibitionof HDAC1 and -2 is insufficient to
reactivate latent HIV-1expression.Reactivation of Latent HIV-1
Expression by Apicidin and
Oxamflatin Is Partially Dependent on the PI3K/Akt
SignalingPathwayPeterlin and co-workers (20) have shown that
SAHAactivates HIV-1 expression in latently infected cells via
thePI3K/Akt pathway. To determine whether this pathway is
alsoactivated by apicidin and oxamflatin, we first assessed
Aktphosphorylation levels in J89GFP cells treated with TNF-
(apositive control) or with 500 nM apicidin, oxamflatin, SAHA,
orscriptaid (Fig. 5A). Increased Akt phosporylation was
observedfollowing treatmentwith apicidin but not oxamflatin, SAHA,
orscriptaid. Because the concentration of SAHA used in
thisexperiment (500 nM) is not sufficient to reactivate latent
HIV-1expression in the J89GFP cells, it was not unexpected that
Aktwas not activated. To further determine the impact of
apicidinand oxamflatin on activation of the PI3K/Akt signaling
path-way, we used PI3K (wortmannin) and Akt (Akt inhibitor IV(AI4))
inhibitors. Indeed, Akt and PI3K inhibitors decreasedbut did not
completely eliminate both apicidin and oxamflatin-induced viral
replication (Fig. 5B). Importantly, these inhibitorshad no
significant effect on the basal levels ofHIV-1 production(Fig. 5B).
These results suggest that the latent HIV-1 reactiva-tion activity
of both apicidin and oxamflatin is intertwinedwithactivation of the
PI3K/Akt signaling pathway.Valproic Acid Is a Weak Inhibitor of
HDAC3In 2004,
Ylisastigui et al. (24) demonstrated that treatment of
restingCD4() T cells of aviremic patients with valproic acid
inducedhistone acetylation and promoted virus outgrowth. An
initialproof-of-concept study in which four volunteers infected
withHIV added oral valproic acid to their cART regimen for 3months
reported a significant decline in the frequency of rest-ing CD()
T-cell infection (25). However, several follow-upstudies found that
valproic acid does not reduce the size oflatent HIV reservoir
(2629). Interestingly, we find that val-proic acid is a weak
inhibitor of the HDAC3-NCOR2 complex(IC50 5.5mM, Table 2) and that
high concentrations of inhib-itor (1 mM) are required in J89GFP
cells to activate HIV-1expression (Fig. 6). The total and free
therapeutic concentra-tions of valproic acid in adults range from
275 to 700 M andfrom 27 to 100 M, respectively. These therapeutic
concentra-tions would be insufficient to inhibit HDAC3 in vivo.
Accord-ingly, our data may explain why valproic acid has no effect
onthe decay of latent HIV reservoirs in patients.ConclusionsThis
study demonstrates that HDAC3 resides
at the HIV-1 LTR in J89GFP cells and that inhibition of thisHDAC
isoform is required for the activation of latent HIV-1expression by
HDACI. Interestingly, Archin et al. (33) recentlydemonstrated that
an HDACI (MRK12) specific for HDAC1
and -2was unable to reactivate latentHIV-1 in J89GFP cells andin
resting CD4() T-cells from aviremic patients. By contrast,an
inhibitor (MRK13) specific for HDAC13 promoted virusoutgrowth in
both assay systems (33). Taken together, thesestudies suggest that
potent inhibition of HDAC3 should be animportant criterion in the
development of HDACI for HIV-1curative strategies. Unfortunately,
neither our study nor that ofArchin et al. (33) could address
whether inhibition of HDAC3alone is sufficient to induce virus
outgrowth or if inhibition ofHDAC1 and/or -2 is also required. The
identification anddevelopment of inhibitors specific forHDAC3may
address thisquestion.Finally, it is likely that there are several
reservoirs of latent
HIV-1 infection in aviremic patients on cART. For
example,resting CD4() T-cells and CD34() multipotent hematopoi-etic
progenitor cells have both been identified as reservoirs oflatent
HIV-1 infection (25). The HDAC isoforms recruited totheHIV-1 LTRs
in these different cell typesmay be different. Inthis regard, the
chemical approach described in this study canreadily be used to
identify the HDAC isoforms that contributeto HIV-1 latency in other
cell types. The primary advantages ofthis approach include the
ability to rapidly conduct studies incell types that cannot be
easily transfected with siRNA (or
FIGURE 5. Activation of the PI3K/Akt signaling pathway by
apicidin andoxamflatin. A, Western blot analysis of Akt
phosphorylation in J89GFP cellstreated with TNF- (positive control)
or with 500 nM apicidin, oxamflatin,SAHA, or scriptaid.
Phosphorylated Akt and -actin were detected usingmonoclonal
antibodies specific for these proteins. B, J89GFP cells weretreated
with 500 nM HDACI with or without wortmannin (100 nM) or the
Aktinhibitor IV (AI 4, 10 M). The number of J89GFP cells expressing
EGFP wasquantitated after 24 h by FACs. Error bars represent S.E.
from three indepen-dent experiments.
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
13
-
shRNA) molecules or in patient-derived tissues or cells
wherethere may be insufficient material to carry out genetic
studies.Importantly, our chemical approach also provides an
immedi-ate assessment of the therapeutic potential of HDACI to
reac-tivate latent HIV-1 expression in different cell types
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FIGURE 6. Reactivation of latent HIV-1 in J89GFP cells by
varying concen-trations (010 mM) of valproic acid. The percentage
of J89GFP cellsexpressing EGFP was quantitated after 24 h by
FACs.
Inhibition of HDAC3 Required for Reactivation of Latent
HIV-1
14
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Host Protein Ku70 Binds and Protects HIV-1 Integrase
fromProteasomal Degradation and Is Required for HIV
Replication*Received for publication, September 13, 2010, and in
revised form, March 28, 2011 Published, JBC Papers in Press,March
29, 2011, DOI 10.1074/jbc.M110.184739
Yingfeng Zheng1, Zhujun Ao2, Binchen Wang, Kallesh Danappa
Jayappa3, and Xiaojian Yao4
From the Laboratory of Molecular Human Retrovirology, Department
of Medical Microbiology, Faculty of Medicine, University
ofManitoba, Winnipeg, Manitoba R3E 0J9, Canada
HIV-1 integrase (IN) is a key viral enzymatic protein actingin
several viral replication steps, including integration. INhas been
shown to be an unstable protein degraded by theN-end rule pathway
through the host ubiquitin-proteasomemachinery. However, it is
still not fully understood how thisviral protein is protected from
the host ubiquitin-proteasomesystem within cells during HIV
replication. In the presentstudy, we provide evidence that the host
protein Ku70 inter-acts with HIV-1 IN and protects it from the
Lys48-linkedpolyubiquitination proteasomal pathway. Moreover, Ku70
isable to down-regulate the overall protein polyubiquitinationlevel
within the host cells and to specifically deubiquitinateIN through
their interaction. Mutagenic studies revealed thatthe C terminus of
IN (residues 230288) is required for INbinding to the N-terminal
part of Ku70 (Ku70(1430)), andtheir interaction is independent of
Ku70/80 heterodimeriza-tion. Finally, knockdown of Ku70 expression
in both virus-producing and target CD4 T cells significantly
disruptedHIV-1 replication and rendered two-long terminal repeat
cir-cles and integration undetectable, indicating that Ku70
isrequired for both the early and the late stages of theHIV-1
lifecycle. Interestingly, Ku70 was incorporated into the
progenyvirus in an IN-dependent way. We proposed that Ku70
mayinteract with IN during viral assembly and accompany HIV-1IN
upon entry into the new target cells, acting to 1) protect INfrom
the host defense system and 2) assist IN integrationactivity.
Overall, this report provides another example of howHIV-1 hijacks
host cellular machinery to protect the virusitself and to
facilitate its replication.
Integration is an obligatory step in the life cycle of all of
theretroviruses and is performed by the viral enzyme integrase
(IN).5 During HIV-1 integration, IN catalyzes the insertion
ofnewly reverse-transcribed 10-kb viral DNA into the hostgenome. In
addition, IN plays important roles in other viralreplication steps,
such as reverse transcription, the nuclearimport of preintegration
complexes (PICs), and chromatin tar-geting. By interaction with the
host chromatin-tethering factorLEDGF/p75, IN preferentially targets
viral DNA into transcrip-tionally active sites in the host genome
to optimize the tran-scription and translation of its gene products
(13). Cellularproteins are recruited to assist IN to accomplish
integrationfrom different pathway, including nuclear import,
shielding INfrom proteasomal degradation, integration site
selection, andgap repair (4). Recently, considerable interest has
been focusedon the functional interaction between IN and host
cellular pro-teins in the hope of disrupting their interactions,
thereby block-ing HIV-1 replication. In an attempt to identify host
cellularpartners for IN, several research groups have identified a
num-ber of IN cofactors using the yeast two-hybrid system,
coimmu-noprecipitation (co-IP) assays, or in vitro reconstitution
of theenzymatic activity of salt-stripped PICs (511). A recent
studyby Studamire et al. (5) found that 12 cellular proteins,
includingKu70, could bind to the INs of both the Moloney murine
leu-kemia virus (MMLV) andHIV-1 through screeningwith a
yeasttwo-hybrid system. However, whether these cellular
cofactorsare associated with HIV-1 IN during HIV replication and
theirfunctional relevance remain unknown.Ku70 is an evolutionarily
conserved protein; it is found ubiq-
uitously in eukaryotes and some prokaryotes, such as Archaeaand
Bacteria (1214). It is well known as a DNA repair proteinand is
part of the nonhomologous end-joining (NHEJ) path-way. Ku70 has
also been implicated in many cellular pro-cesses, including
antigen-receptor gene rearrangement,mobilegenetic element biology,
V(D)J recombination of immunoglob-ulins, telomere maintenance, DNA
replication, transcription,cell cycle control, and apoptosis (13,
15). As a DNA repair pro-tein, Ku70 can bind to any double-stranded
DNA irrespectiveof sequence specificity or end configuration,
including 5 over-hangs, 3 overhangs, or blunt ends (for a review,
see Ref. 15).Ku70 can also bind specific DNA sequences to affect
gene tran-scription (16). Formost biological functions inwhichKu70
par-ticipates, Ku functions as a heterodimer consisting of Ku70
andKu80, named according to their respectivemolecular masses of
* This work was supported by Canadian Institutes of Health
Research(CIHR) Grants HOP-81180 and HBF 103212 and the Leaders
OpportunityFund Award from the Canadian Foundation of Innovation
(to X.-J. Y.).
1 Recipient of studentships from the Manitoba Health Research
Council/Manitoba Institute of Child Health (MHRC/MICH) and the CIHR
Interna-tional Infectious Disease and Global Health Training
Program.
2 Recipient of a postdoctoral fellowship from the CIHR
International Infec-tious Disease and Global Health Training
Program.
3 Recipient of studentships from MHRC/MICH.4 Recipient of the
Basic Science Career Development Research Award from
the Manitoba Medical Service Foundation. To whom
correspondenceshould be addressed: Laboratory ofMolecular
HumanRetrovirology, Dept.ofMedicalMicrobiology, Faculty
ofMedicine,University ofManitoba, 508745 William Ave., Winnipeg R3E
0J9, Canada. Tel.: 204-977-5677; Fax: 204-789-3926; E-mail:
[email protected].
5 The abbreviations used are: IN, integrase; PIC, preintegration
complex; IP,immunoprecipitation;WB,Westernblot;MMLV,Moloneymurine
leukemiavirus; NHEJ, nonhomologous end-joining; Ub, ubiquitin;
VSV-G, vesicularstomatitis virus G; p.i., postinfection; PL,
ProLabel; aa, amino acids; MOI,multiplicity of infection.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp.
1772217735, May 20, 2011 2011 by The American Society for
Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
15
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70 and 80 kDa. Two regions of Ku70 amino acids 1115 and430482
are responsible for its heterodimerization with Ku80(17).
Successful HIV-1 integration requires gap repair betweenviral DNA
and host genome, which is believed to be performedby host DNA
repair enzymes (18). Two different host DNArepair pathways have
been suggested to fill in the gap duringHIV-1 infection: theNHEJ
andDNAdamage-sensing pathways(1921). TheNHEJ pathway begins with
the recruitment of theKu70/80 heterodimer, followed by the
catalytic subunit ofDNA-dependent protein kinase or DNA-PKcs,
Xrcc4, andDNA ligase IV. Studies have shown that the NHEJ pathway
isimportant for retroviral transduction or infection and for
thecell survival of infected or transduced cells (20, 2225).
Forexample, HIV-1-based vector transduction or infection
wasmarkedly reduced in cells deficient in Ku80, DNA-PKcs, Xrcc4,or
ligase IV (22, 24). Moreover, NHEJ activity is required fortwo-long
terminal repeat (2-LTR) circle formation, and Ku70has been detected
in MMLV PICs (24, 2628). Ku80 was alsoshown to suppress HIV
transcription by specifically binding toa negative regulatory
element within the LTR (29). All of theseobservations suggest that
Ku70 or the K70/80 heterodimermaybe involved inHIV-1 infection by
affectingmultiple steps of theviral replication cycle, such as
integration. In addition, a noveldeubiquitinating enzymatic
activity of Ku70 was recentlydescribed in which Ku70 has a
regulatory effect on Bax-medi-ated apoptosis by decreasing the
ubiquitination of Bax andblocking Bax from proteasomal degradation
(30). However,whether Ku70 also exerts a deubiquitinating effect on
otheridentified binding partners of Ku70 and how Ku70 interactswith
the ubiquitin-proteasome pathway to deubiquitinate pro-tein
substrates are still unclear.In this study, we investigated the
interaction between Ku70
and HIV-1 IN and the potential roles of Ku70 during
HIV-1replication using cell-based coimmunoprecipitation and
shorthairpin RNA (shRNA)-mediated knockdown
approaches.Interestingly, our results provide evidence that Ku70 is
able toprotect HIV-1 IN from Lys48-linked polyubiquitination
anddegradation by down-regulation of the overall protein
poly-ubiquitination level within the host cells and by specific
INdeubiquitination through its binding to IN. Moreover, ourstudy
showed that Ku70 depletion in both virus-producing andtarget cells
drastically inhibited HIV-1 replication and blocked2-LTR formation
and integration in the real-time PCR analysis.Our data also showed
that, mediated by HIV-1 IN, Ku70 wasincorporated into the progeny
virus. All of these results suggestthat Ku70 may interact with IN
during viral assembly andaccompany HIV-1 IN into newly infected
cells to assist INintegration activity and protect IN from
host-mediateddegradation.
EXPERIMENTAL PROCEDURES
Cell Lines and TransfectionHuman embryonic kidney293T and HeLa
cell lines were cultured in Dulbeccos modifiedEagles medium (DMEM)
supplemented with 10% fetal calfserum (FCS) and 1%
penicillin/streptomycin. Human CD4C8166 T-lymphoid cells were
maintained in RPMI 1640medium supplemented with 10% FCS and 1%
penicillin/strep-tomycin. For the transfection of 293T cells and
HeLa cells, the
standard calcium phosphate precipitation technique was used,as
described previously (31).Plasmids and ReagentsTo achieve high
level IN expression,
a codon-optimized IN (INopt) cDNA was synthesized andcloned into
the pUC57 vector (GenScript Co., Ltd.). To con-struct pAcGFP-INopt,
the INopt fragment was excised frompUC57-INopt with BamHI and
cloned in frame at the 3 end ofthe pAcGFP1-C vector (Clontech) with
the same restrictionenzyme. To construct pAcGFP-INwt/mut, each of
the INwt/mut coding sequences, including 1230, 1250, 1270,50288,
112288, and K186A/R187A, was amplified by PCR-based mutagenesis and
subcloned into the pAcGFP1-C vector(Clontech) in frame with the GFP
coding sequence at the BglIIand BamHI restriction sites (32).
Plasmids IN-YFP and MA-YFP were described previously (33, 34).
Untagged human full-length Ku70 cDNA in the pCMV6-XL5 vector was
purchasedfrom OriGene Technologies Inc. To construct SVCMV-T7-Ku70,
a Ku70 cDNA without a start codon was amplified andcloned into the
SVCMVin-T7 vector at the BamHI and NotIrestriction sites. The
T7-Ku70 truncation mutants (1263,1430, 226609, and 430609) were
obtained using the samestrategy. The nucleotide sequences of the
mutagenic oligonu-cleotides are as follows: 5Ku70-BamHI,
5-TAGCCGGATCC-TCAGGGTGGGAGTCATATTA-3; 3Ku70-NotI,
5-TATAT-GCGGCCGCTCAGTCCTGGAAGTGCTT-3;
Ku70263-NotI,5-AGATGCGGCCGCCTAGAGCTTCAGCTTT-3; Ku70430-NotI,
5-TATGCGGCCGCTCATGGAGGAGTCACCT-GAAT-3; Ku70226-BamHI,
5-TATGGATCCGATGAGGA-CCTCA-3; Ku70430-BamHI,
5-ATATGGATCCCCAGGC-TTCCAGCT-3. HA-tagged ubiquitin (HA-Ub) and
mutantsHA-UbK48R and HA-UbK63R were described previously
(35).TheHIV-1 proviruses pNL4.3-GFP,HxBru, andHxBru-IN-HAwere
described earlier (32, 36). For single cycle HIV virus,RT/IN/Env
gene-deleted NL4.3lucBglRI provirus andCMV-Vpr-RT-INwere described
previously (34, 37, 38). CMV-Vpr-RT has two stop codons TAGTGA in
place of the first sixnucleotides in IN sequences, and the sequence
was confirmedby sequencing. To construct CMV-Vpr-RT-IN-ProLabel
(Vpr-RT-IN-PL) plasmid, a two-step-based PCR method was
used.ProLabel tag sequence was amplified from the pProLabel-Cvector
from ProLabelTM Detection Kit II (Clontech) andinserted after the
IN sequence in the CMV-Vpr-RT-IN plasmidwith the IN stop codon and
ProLabel start codon removed. Thefollowing primers were used: RT
NheI 5, 5-GCAGCTAGCA-GGGAGACTAA-3; R-RT-IN-ProLabel pstI 3,
5-GTCGACT-GCAGAATTCGAAGCTTATTC-3; IN-ProLabel 5,
5-AGA-CAGGATGAGGATAGCTCCAATTCACTG-3; IN-ProLabel3,
5-CAGTGAATTGGAGCTATCCTCATCCTGTCT-3.Antibodies and ReagentsThe
rabbit anti-GFP polyclonal
antibody (Molecular Probes Inc.), the rabbit anti-HA
antibody(Sigma), and mouse anti-T7 antibody (Novagen) were usedfor
immunoprecipitation. The antibodies for Western blot(WB) were as
follows: themousemonoclonal anti--actin anti-body (Abcam), mouse
anti--tubulin (Sigma), mouse anti-Ku70 (Abcam), mouse anti-Ku80
(Abcam), horseradishperoxidase (HRP)-conjugated anti-GFP antibody
(MolecularProbes), HRP-conjugated anti-HA antibody (Miltenyi
Biotec),and HRP-conjugated anti-T7 antibody (Novagen). As the
sec-
Ku70 Binds and Protects HIV-1 Integrase
16
-
ondary antibodies, the ECLTM HRP-conjugated donkey anti-rabbit
IgG and sheep anti-mouse IgG were purchased fromAmersham
Biosciences. The WB detection ECL kit was pur-chased from
PerkinElmer Life Sciences (Boston, MA). NonidetP-40 was from Roche
Applied Science. Proteasome inhibitorMG-132 and puromycin were
obtained fromCalbiochem. Sub-tilisin was purchased from
Sigma.Transient and Stable Knockdown of Ku70 in 293T, HeLa
Cells, and C8166 T CellsTo test the effect of Ku70 levels onthe
stability of IN, siRNA targeting human Ku70 (GenBankTMaccession
number NM_001469) was used to transiently knockdown Ku70 expression
in 293T cells and HeLa cells using theLipofectamineTM RNAiMAX
transfection reagent (Invitro-gen). The sense primer for this siRNA
is 5-GAUCCAGGUU-UGAUGCUCAtt-3, targeting Ku70 nucleotides
10941112.In parallel, a scrambled siRNA (Invitrogen) was used as a
neg-ative control (siNC). After 5 nM siKu70 or siNC
oligonucleotidewas transfected into cells for 12 h, cells were
transfected againwith 5 nM siKu70 to maximize knockdown
efficiency.To produce a stable Ku70-knockdown (KD) 293T and
C8166 CD4 T cell line, lentivirus-like particles harboringKu70
shRNA were produced by cotransfecting the shRNApLKO.1 vector
containing shRNA targeting the Ku70
mRNA(5-CCGGCGACATAAGTCGAGGGACTTTCTCGAGA-AAGTCCCTCGACTTATGTCGTTTTTG-3
(Oligo ID:TRCN0000039608; purchased from Open
Biosystems)),packaging plasmid 8.2, and vesicular stomatitis virus
G(VSV-G) expressor into the 293T cells. After 48 h, shRNApLKO.1
vector particles were pelleted by ultracentrifugation(32,000 rpm at
4 C for 1 h) and used to transduce cells for48 h, followed by
selection with 2 g/ml puromycin for 1week. Ku70-KD efficiency was
determined by WB analysiswith anti-Ku70 antibody. Endogenous -actin
was used tonormalize sample loading. The pLKO.1 vector without
theshRNA sequence (empty vector) was introduced into cells bythe
same method as a negative control.Direct Immunofluorescence AssayTo
test the effect of
Ku70-KD level on the expression of IN, HeLa cells were
firsttransfected with Ku70-specific siRNA oligonucleotides or
non-targeting random siRNA (siNC) for 48 h and further trans-fected
with GFP-INopt for another 48 h with or withoutMG-132 (10 M)
treatment. GFP fluorescence-positive cellswere imaged by microscopy
under a 20 objective lens (CarlZeiss).Coimmunoprecipitation Assay
in 293T Cells and in HIV-1-
infected C8166T CellsTo detect the interaction
betweenGFP-INwt/mut and T7-Ku70wt/mut and to identify theirmutual
binding regions, the cell-based co-IP assay was per-formed as
described previously (37). Briefly, GFP orGFP-INwt/mut plasmid was
cotransfected with pCMV-Ku70 or T7-Ku70,respectively, into 293T
cells for 48 h. To increase GFP-IN sta-bility, 10MMG-132was added
12 h prior to cell lysis for co-IP.Then 90% of the transfected
cells were lysed in 0.25% NonidetP-40 prepared in 199medium
containing a mixture of proteaseinhibitors (Roche Applied Science)
and clarified by centrifuga-tion at 14,000 rpm for 30 min at 4 C.
Supernatant was pre-cleared with Protein G-agarose on a rotator for
2 h at 4 C andsubsequently subjected to IP with a rabbit anti-GFP
antibody
and Protein A-Sepharose overnight. The IN-bound proteinswere
detected by WB using anti-Ku70 or anti-T7 antibodies.The same
nitrocellulose membrane was then stripped andprobed with anti-GFP
antibody to detect GFP-INwt/mut orGFP expression. Meanwhile, 5% of
the transfected cells werelysed in 0.5% Nonidet P-40, and the
lysates were used to detectthe expression of GFP-INwt/mut and Ku70
by WB using theircorresponding antibodies.To examine the IN/Ku70
interaction inHIV-1-infected cells,
HIV-1 (HxBru or HxBru-IN-HA)-infected C8166 T cells werelysed
with 0.25% Nonidet P-40 and immunoprecipitated withanti-HA antibody
followed by WB with anti-Ku70 antibody todetect IN-bound
Ku70.Detection ofUbiquitination of IN in theAbsence or Presence
of
Ku70To determine the ubiquitination level of HIV-1 IN inthe
absence and presence of Ku70, 293T cells were
cotrans-fectedwithGFP-IN andHA-Ubwild type ormutants K48R andK63R
with and without T7-Ku70wt or T7-Ku70(1430). After48 h, cells were
lysed in 199 medium containing 0.25%NonidetP-40 and a
protease/inhibitor mixture and immunoprecipi-tatedwith anti-GFP
antibody. Then the precipitated complexeswere run on a 10%
SDS-polyacrylamide gel and analyzed for thepresence of HA-Ub
byWBwithHRP-conjugated anti-HA anti-body. Simultaneously, GFP-IN
was detected by immunoblot-ting the same membrane with
HRP-conjugated anti-GFP anti-body.Andprotein band intensitywas
quantified usingQuantityOne 1-D analysis software (Bio-Rad).Virus
Production and InfectionTo study the effect of
Ku70-KD on HIV-1 replication, equal amounts (quantified byHIV-1
p24 antigen) of pNL4.3-GFP virus were used to infectKu70-KD or
empty vector-transduced C8166 T cells for 2 h;cells were then
washed and cultured in a 37 C incubator. Atdifferent time points,
viral replication levels weremonitored bythemeasurement of p24
levels using an HIV-1 Gag-p24 ELISA.To test the infectivity of
progeny virus produced from theKu70-KD cells, empty-vector and
Ku70-KDC8166 T cells wereinfected with the same amounts of
pNL4.3-GFP. Progenyviruses were collected by ultracentrifugation
after 4 days ofinfection, and equal amounts of viruses (quantified
by HIV-1p24 antigen) were used to infect empty vector or
Ku70-KDC8166 T cells. Viral infection was examined at 3 days
postin-fection by monitoring HIV p24 levels in the
supernatant.Quantitative Real-time PCR1.5 106 stable C8166 T
cell
lines with Ku70-KD or empty vector-transduced were infectedwith
the pNL4.3-GFP virus as described above. Heat-inacti-vated virus
(70 C for 30min) was used as a negative control forinfection. After
4 h of infection, cells were washed and culturedin fresh RPMI
medium. At 24 h postinfection, cells were har-vested and washed
with PBS twice. DNA was isolated using aQIAamp blood DNA minikit
(Qiagen). The total levels ofHIV-1 DNA, 2-LTR circles, and
integrated DNA were quanti-fied following the same procedure in an
Mx3000P real-timePCR system (Stratagene) as described (32).Virus
Composition and Incorporation of Cellular Protein into
HIV-1 VirionTo examine the viral protein compositions,
thepNL4.3-GFP viruses from empty vector-transduced andKu70-KD C8166
T cells were pelleted through a 20% sucrosecushion at 35,000 rpm
for 1.5 h at 4 C. Then equal amounts of
Ku70 Binds and Protects HIV-1 Integrase
17
-
viruses (normalized by p24 values) were lysedwith 4Laemmlibuffer
and directly loaded onto an SDS-PAGE gel and analyzedfor IN and p24
expressions using their corresponding antibod-ies. The reverse
transcription activity from the purified viruseswas analyzed by a
reverse transcription assay using a commer-cial RT assay kit
(RocheApplied Science) according to theman-ufacturers
instructions.To detect the presence of Ku70 in the HIV-1 particles,
15
106 CD4 C8166 T cells were mock-infected or infected
withpNL4.3-GFP for 3 days. Then supernatants from both cell
cul-tures were ultracentrifuged at 35,000 rpm for 1.5 h through
a20% sucrose cushion. The pellets were dissolved in the samevolume
of radioimmune precipitation assay buffer and mixedwith 20% (v/v)
TCA, followed by precipitation on ice for 30minand acetone washing.
Protein precipitates were dissolved in 4Laemmli buffer and directly
loaded onto a 10% SDS-polyacryl-amide gel. Virus-associated Ku70
and p24 were then examinedby WB using the corresponding
antibodies.Subtilisin treatment of purified HIV-1 virions. The
subtilisin
assay was performed according to the protocol as described(39).
The Vpr-RT-IN or Vpr-RT expressor was cotransfectedwith VSV-G
andNL4.3lucBglRI to produce single cycle INand IN virus. The
viruses were first ultracentrifuged through20% sucrose at 35,000
rpm for 2 h and then mock-treated ortreated with 0.1 mg/ml of
subtilisin (Sigma) for 20 h at a 37 Cincubation. Subtilisin was
inactivated by phenylmethylsulfonylfluoride. Virus was then
repelleted as described above, lysed inradioimmune precipitation
assay buffer, and loaded onto SDS-polyacrylamide gel followed by
WB. Blots were sequentiallyprobed with anti-Ku70, anti-IN, and p24
antibodies.ProLabel Detection AssayTo test the effect of Ku70 on
IN
during HIV infection, VSV-G pseudotyped HIV single cyclevirus
containing ProLabel tag fused to the C terminus of INwasgenerated
to quantify IN expression under HIV infection.NL4.3lucBglRI was
cotransfected with Vpr-RT-IN-PL andVSV-G expressor into 293T cells
to generate VSV-G pseu-dotyped HIV-1 single cycle IN-PL virus. The
viruses were usedto infect shKu70-KD or empty vector-transduced
C8166T cellsfor 3 h. The cells were washed three times and kept in
freshmedium and then lysed with lysis/complementation buffer at8 h
p.i. IN-ProLabel activity in the cell lysate was measuredaccording
to the manufacturers instructions from the assay kit(ProLabelTM
detection kit II, Clontech).Statistical AnalysisThe statistical
significance was calcu-
lated using Students t test, and a p value of0.05 was
consid-ered significant.
RESULTS
Cellular Protein Ku70 Protects HIV-1 IN from
ProteasomalDegradationAs a part of the NHEJ machinery, the host
pro-tein Ku70 has been shown to participate inHIV integration andin
the circularization of unintegrated viral DNAs (25, 27).
Sur-prisingly, based on the results of a yeast two-hybrid assay,
arecent study indicated that HIV-1 IN may bind to Ku70
(5),suggesting a direct association betweenHIV-1 IN andKu70.
Tofurther investigate this viral/host protein interaction, we
coex-pressed Ku70 (T7-tagged Ku70) and HIV-1 IN (IN-YFP) in293T
cells and analyzed their interaction after 48 h of transfec-
tion. Noticeably, our results revealed that T7-Ku70
overexpres-sion significantly increased IN expression (Fig. 1A,
lanes 1 and2).However, the coexpression ofKu70with anotherHIV-1
pro-tein, MA (MA-YFP), did not change the MA expression level(Fig.
1A, lanes 3 and 4). This suggests that Ku70 is able toincrease IN
expression. Alternatively, Ku70 could protect theIN protein from
degradation (40).To further test whether endogenous Ku70 could
exert the
same activity andwhether it is due to a protective effect, we
firstknocked down the Ku70 expression using specific siRNA in293T
(Fig. 1B) or HeLa cells (Fig. 1C) and checked the level ofGFP-IN
expression by WB or fluorescence microscopy (Fig. 1,B and C). To
increase IN expression under normal conditions,we used a pAcGFP-IN
with a codon-optimized IN sequence(GFP-INopt). The results showed
that IN expression inKu70-KD cells was significantly decreased when
comparedwith IN expression in siNC cells (Fig. 1, B (compare lanes
1 and2) and C (compare A1A3 and B1B3)). Intriguingly, in
thepresence of the specific proteasome inhibitorMG-132 (10M),IN
expression in Ku70-KD cells was remarkably increased,reaching
levels similar to those in siNC-transfected 293T andHeLa cells
(Fig. 1, B (compare lanes 4 a