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

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

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

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

&

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.

V

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

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