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THSE PRSENTE
POUR OBTENIR LE GRADE DE
DOCTEUR DE BIOCHEMIE
COLE DOCTORALE SCIENCE DE LA VIE
BIOCHEMIE
Jane Etegeneng BESONG
MOLECULAR INSIGHTS INTO A PUTATIVE POTYVIRUS RNA ENCAPSIDATION
PATHWAY AND POTYVIRUS
PARTICLES AS ENZYME NANO-CARRIERS
Sous la direction de, Thierry MICHONCo-directeur, Kristiina
MKINEN
Soutenue le 14 Juin 2016
Membres du jury
M. TEERI Teemu, Principal Investigator, Senior Scientist,
University of Helsinki, Finland, PrsidentMme. WEGE Christina,
Professeur, Universitt Stuttgart, Deutschland, RapporteurM. HYTONEN
Vesa, Group Leader, University of Tampere, Finland, Rapporteur
M. TALIANSKY Michael, Honorary Lecturer, University of Dundee,
Scotland, RapporteurMme MKINEN Kristiina, Group Leader, University
of Helsinki, Finland, Directrice de thse
M. MICHON Thierry,Directeur de Recherche INRA, Universit de
Bordeaux, Directeur de thse
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Molecular insights into a putative RNA encapsidation pathway and
potyvirus particles as enzyme nano-carriers
Rsum
La prsente tude avait pour but d'identifier de nouvelles
stratgies pour la prsentation slective d'enzymes la surface de
application potentielle dans la technologie des biocapteurs ou
des puces protines. Les potyvirus ont t choisis comme nano-supports
modles. Les Potyvirus, le genre le plus large de la famille des
Potyviridae, la seconde plus grande famille de virus de plante,
sont responsables de trs graves pertes dans les cultures. Ils
forment des capsides flexibles en forme de btonnet entourant une
seule molcule d'ARN positif simple brin. Les vnements molculaires
conduisant la slection et l'encapsidation spcifiques de l'ARN
potyviral sont inconnus. Afin de mieux exploiter le potentiel de
ces virus comme nano-supports, la premire tape de ce travail a
d'encapsidation de l'ARN de particules de potyvirus. Des tudes
prcdentes ont montr que la protine d'enveloppe (CP) du virus de la
pomme de terre A (PVA) interfre avec la traduction de l'ARN viral
lorsqu'elle est fournie en excs en trans suggrant que cela pourrait
se produire pour
cette tude, nous avons montr que cette inhibition est mdie par
des interactions CP-CP co-traductionnelles se produisant entre deux
populations de CP, produites en
trans et en cis et permettant trs probablement le recrutement
spcifique de l'ARN potyviral pour son encapsidation. En accord avec
les tudes d'assemblage in vitro publies prcdemment nous proposons
un
l'ARN viral est initie par des interactions CP-CP
co-traductionnelles. Dans la deuxime partie de ce travail,
diffrentes approches ont tdes enzymes sur les platesformes
virales
intermdiaires ractionnels. Parmi les trois stratgies testes
seule celle utilisant un peptide qui se liant aux anticorps, le
peptide z33 de la protine A de Staphylococcus aureus a t couronne
de succs. Une couverture de 87 % des sites sur les particules de
potyvirus avec l'enzyme a t obtenue. Cette stratgie a t utilise
pour piger deux enzymes, la 4-coumarate: coenzyme A ligase (4Cl2)
et stilbne synthase (STS), catalysant des tapes conscutives dans la
voie de synthse de resvratrol partir de lysats cellulaires
particules de potyvirus immobilises sur les parois d'un tube en
polypropylne. Cette stratgie rassemble les approches ascendante et
descendante pour construire des nanomatriaux base de virus et offre
un moyen efficace et conomique pour co-immobiliser et purifier des
enzymes
Mots cls
nano-supports, enzymes, encapsidation, immobilisation,
nano-technologie, particules virales
II
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Molecular insights into a putative RNA encapsidation pathway and
potyvirus particles as enzyme nano-carriers
Abstract
The present study intended to identify newstrategies for the
selective presentation ofbiocatalysts on the surface of
viralnanoparticles with potential application inbiosensor
technology or protein chips.Potyviruses were chosen as model
nanoscaffolds for biocatalysts. Potyviruses are the largest genus
in the family Potyviridae and cause significant plant damage. They
form flexible rod-shaped capsids surrounding a single stranded
positive sense RNA molecule. The molecular events leading to the
specific selection and encapsidation of potyviral RNA are unknown.
To better exploit the potential of these viruses as
nanocarriers,the first step in this study was to look into their in
vivo RNA encapsidation process.Earlier studies showed that Potato
virus A(PVA) coat protein (CP) interferes with viral RNA
translation when provided in excess in trans and it was suggested
this could occur to initiate viral RNA encapsidation. In this
follow up study, we used the agroinfiltration approach for the
transient expression of full length, truncated or mutated viral
RNAs with wild type CP (CPwt) and showed that this inhibition is
mediated by co-translational CPCPinteractions occurring between two
CPpopulations, produced in trans and in cis.Because CP inhibited
translation of the entire viral genome and virus particles were
formed later than during normal infection, it was assumed that the
CP acted during thisinhibition process to specifically recruit
viral RNA for encapsidation. In line withpreviously published in
vitro assemblystudies, we propose a mechanism throughwhich viral
RNA encapsidation is initiatedthrough co-translational CP-CP
interactions. The second part of this work entailed the
investigation of novel approaches for organizing biocatalysts on
virus platforms. The aim was to control the display of enzymes on
virus surfaces whilemaximizing channelling of reaction
intermediates. Three strategies were tested but only one involving
an antibody binding peptide, the z33 peptide from Staphylococcus
aureus was successful. An 87 % occupancy of accessible sites on
thepotyvirus particles by the enzyme wasachieved. The same strategy
was used to graft potyvirus particles with two enzymes: 4-
coumarate:coenzyme A ligase (4CL2) and stilbene synthase (STS),
catalysingconsecutive steps in resveratrol syntheticpathway or a
protein chimera, generated bythe genetic fusion of both enzymes.
This was achieved by trapping either the monoenzymes or the protein
chimera fromclarified soluble E. coli cell lysates on to thesurface
of potyvirus particles pre-immobilized in a polypropylene
tube.Resveratrol was synthesized from bothmono-enzymes and the
protein chimera insolution and on potyvirus particles. Thisstrategy
brings together a bottom-up and topdown approach for designing
virus basednano-materials and offers a cost effective and efficient
way to co-immobilize and purify enzymes.
Keywords
nano-carriers, enzymes, encapsidation, immobilisation,
nano-technology, virus particles
III
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Unit de recherche
Division of Microbiology and BiotechnologyDepartment of Food and
Environmental Sciences
Faculty of Agriculture and ForestryP.O. Box 56 (Viikinkaari
1)
FI - 00014 University of HelsinkiFinland
Equipe de VirologieUMR 1332 Biologie du Fruit et Pathologie
INRA et Universit de BordeauxCS 20032
33882 Villenave d'Ornon CedexFrance
IV
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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications,
referred to by their roman numerals throughout the text.
I. Pille J, Cardinale D, Carette N, Di Primo C, Besong-Ndika J,
Walter J, Lecoq H, van Eldijk MB, Smits FC, Schoffelen S, van Hest
JC, Mkinen K, Michon T. General strategy for ordered noncovalent
protein assembly on well-defined nanoscaffolds. Biomacromolecules.
2013 Dec 9;14 (12):4351-9. doi: 10.1021/bm401291u
II. Besong-Ndika J, Ivanov KI, Hafrn A, Michon T, Mkinen K.
Cotranslational coat protein-mediated inhibition of potyviral RNA
translation. J Virol. 2015 Apr;89 (8):4237-48. doi:
10.1128/JVI.02915-14
III. Besong-Ndika, J., Wahlsten, M., Cardinale, D., Pille, J.,
Walter, J., Michon, T., and Mkinen, K. Toward the Reconstitution of
a Two-Enzyme Cascade for Resveratrol Synthesis on Potyvirus
Particles. 2016. Front. Plant Sci. DOI:10.3389/FPLS.2016.00089
UTION
I. Jane Besong-Ndika participated in the research work, analysis
and data interpretation.
II. Jane Besong-Ndika designed and executed most of the
experimental work with the exception of RNA gel shift experiments.
She analysed the results and interpreted the results with
co-authors. JBN wrote the first draft of the manuscript.
III. Jane Besong-Ndika designed and executed most of the
experiments except for the MS analysis. She interpreted the results
together with co-authors and wrote the first draft of the
manuscript.
Unpublished data is also presented which showcase the work JBN
carried out to identify a successful strategy to graft functional
biological molecules on potyvirus scaffolds.
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VII
TABLE TO CONTENTS
ABBREVIATIONS IX
INTRODUCTION 11
Section 1: Viruses as Nanoparticles 11
1. Definition of viral nanoparticles (VNPs) 112. Why plant
viruses? 113. Architecture of VNPs 12
3.1. Icosahedral VNPs 123.2. Rod-shaped VNPs 133.3. RNA-driven
viral architectures 14
4. Production of VNPs 144.1. Native VPs 144.2. VLPs 16
5. Functionalization of VNPs 165.1. Covalent attachment by
bioconjugation 165.2. Genetic engineering of the capsid protein
175.3. Non-covalent strategies 18
5.3.1. Bio-conjugation via streptavidin-biotin interaction
185.3.2. Utilization of coiled coils for functionalization 18
5.4. Other functionalization strategies 186. Application of VNPs
19
6.1. Nanomedicine 196.2. Nano-electronics 206.3. Modern
enzymology 20
Section 2: Potyviruses as nano-platforms 20
7. Potyviruses 208. Structure of Potyvirus CP and virions 219.
Potyvirus assembly 2210. Potyvirus VLP production 2311. Application
of potyviruses 23
11.1. Biotechnology 2311.2. Nanotechnology 23
Section 3: Multi-enzyme systems and Immobilization 24
AIMS OF THE STUDY 25
SUMMARY OF MATERIALS AND METHODS 26
SUMMARY OF RESULTS AND DISCUSSION 29
Section 1: Insights into the assembly process of PVA particles
(II) 29
1. Dose-dependent inhibition of translation 29
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VII
2. Translation inhibition occurs via the CP mRNA 293.
Co-translational CP-CP interactions inhibit viral RNA translation
304. Possible regulation of the shift from viral RNA translation to
encapsidation by CP 31
Section 2: Building functionalized potyvirus-based nano-carriers
32
5. Via IgG binding peptide, z33 (I,II) 335.1. Expression of
z33-tagged enzymes 335.2. z33-IgG parameters 345.3. Macromolecular
assembly in solution and on carbon coated grids 345.4. Potyvirus
particles as multi-enzyme nano-carriers 35
6. Leucine zipper strategy (unpublished results) 367.
PVA-binding peptide strategy (unpublished results) 38
CONCLUDING REMARKS AND PERSPECTIVES 40
ACKNOWLEDGEMENTS 42
REFERENCES 44