To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP), and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and cyanophage. When analysed statistically, patterns of virus abundance suggested that the seasonality of any one virus cannot be generalised to predict that of other viruses, even among closely related viruses. This study also demonstrated a strong relationship between algal virus abundance and host biomass. It was found that despite the apparent heterogeneity of virus abundance across the Bay, virus abundance patterns clustered by sampling date and geographic location. By providing evidence for diverse algal viruses with complex seasonality, this work highlights significant gaps in the current understanding of Bay of Quinte phytoplankton ecology.

Most existing chemotherapeutics lack adequate specificity for transformed cells and therefore have high rates of collateral damage to normal tissue. Moreover, such therapies often depend on p53 to induce cell death and are ineffective on the large number of human cancers that have lost p53 function. The discovery of novel p53-independent cancer therapies is therefore of significant interest. The Chicken Anemia Virus protein Apoptin selectively induces apoptosis in transformed cells in a p53-independent manner while leaving normal primary cells unaffected. This selectivity is thought to be largely due to cell type-specific localization: in primary cells Apoptin is cytoplasmic, whereas in transformed cells the protein localizes to the nucleus. The basis for this cell type-specific localization remains to be determined. In this study, Apoptin is revealed to be a nucleo-cytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, as Apoptin fragments containing either the NES or NLS fail to localize differently between transformed and primary cells. Significantly, cell type-specific localization can be rescued in trans by co-expression of the two separate fragments, which are able to interact through an Apoptin multimerization domain. Interestingly, this multimerization domain overlaps with the NES suggesting that these two activities may be functionally coupled in cytoplasmic retention in primary cell types. Factors present in transformed cells induce localization of Apoptin to the nucleus where a biochemically distinct, more soluble form of the protein exists. Using affinity-purification and mass spectroscopy it was found that, specifically in transformed cells, Apoptin is associated with APC1, a subunit of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is required to establish a mitotic cell-cycle checkpoint, and its inhibition results in G2/M arrest and apoptosis. Expression of wild type Apoptin in transformed cells inhibits APC/C function and induces G2/M arrest and apoptosis, whereas Apoptin mutants that are unable to associate with APC1 have no effect. In p53 null cells, ablation of APC1 by RNA interference induces a G2/M arrest and apoptosis analogous to that observed following Apoptin expression. Furthermore, Apoptin was found to induce the formation of PML bodies and to recruit APC/C subunits to these nuclear structures suggesting a mechanism involving sequestration and subsequent inhibition of the APC/C. Thus, the results of this study clarify Apoptin cell type-specific localization behavior and explain the ability of Apoptin to induce apoptosis in transformed cells in the absence of p53. This study advances a newly emerging field of viral mechanisms of apoptosis involving G2/M arrest and APC/C modulation. The resultant p53-independent apoptosis suggests that the APC/C may be an attractive target for the development of anti-cancer drugs.

Les rétrovirus sont un enjeu de santé publique, aussi bien humaine qu'animale. La compréhension des déterminants structuraux sous-jacents à la fonction de leurs protéines constitue une étape essentielle dans le développement de stratégies antirétrovirales efficaces.Ce manuscrit porte sur l'étude des bases structurales des mécanismes moléculaires impliqués dans les fonctions clés des rétrovirus que sont i) la régulation de l'expression des protéines de rétrovirus complexes et ii) l'assemblage des particules virales, à travers l'étude de deux protéines rétrovirales d'intérêt thérapeutique : la protéine Tax du virus T-lymphotrope humain (HTLV) et la protéine de capside du virus de l'immunodéficience féline (FIV). L'étude structurale de ces deux protéines d'intérêt et la compréhension des mécanismes moléculaires nécessaires à leurs fonctions permettraient d'ouvrir la voie à la conception de nouvelles stratégies antirétrovirales.Malgré de nombreux tests d'expression et de purification, l'étude structurale de la protéine Tax du HTLV n'a pu être réalisée, en raison de son insolubité. Cependant, ce travail doctoral a permis de résoudre, pour la première fois, la structure cristallographique de la protéine de capside entière du FIV. Bien que cette dernière adopte un repliement similaire aux autres capsides rétrovirales dont la structure est connue, elle présente également des spécificités structurales dont les conséquences fonctionnelles seront discutées / Retroviruses are a major concern of public health in humans but also in animals. A better understanding of the structural determinants underlying the functions of retroviral proteins is a crucial step for the development of efficient antiretroviral therapies.This manuscript studies the structural basis of the molecular mechanisms implicated in key functions of retroviruses such as, i) the regulation of complex retroviruses protein expression and ii) the assembly of viral particles, through the study of two retroviral proteins of therapeutic interest: the human T-lymphotropic virus (HTLV) Tax protein and the feline immunodeficiency virus (FIV) capsid protein. The functional and structural studies of these two proteins and the understanding of the molecular mechanisms required for their functions will pave the way to the conception of new antiretroviral therapeutic strategies.Despite several expression and purification assays, no structural studies could be performed for the HLTV Tax protein. However, this study allowed the resolution of the first structure for the full-length FIV capsid protein by X-ray crystallography. Although the FIV capsid protein displays a standard a-helical topology like other retroviral CAs, it also harbors original features whose functional consequences will be discussed

Cette thèse propose de nouveaux outils pour la caractérisation locale de familles de protéines au niveau de la séquence et de la structure. Nous introduisons les fragments en contact (CF) comme des portions de structure conciliant localité spatiale et voisinage séquentiel. Nous montrons qu'ils bénéficient d'une meilleure prédictibilité de structure depuis la séquence que des fragments contigus ou encore que des paires de fragments qui ne seraient pas en contact en structure. Pour comparer structuralement ces CF, nous introduisons l'ASD, une nouvelle mesure de similarité ne nécessitant pas d'alignement préalable, respectant l'inégalité triangulaire tout en étant tolérante aux décalages de séquences et aux indels. Nous montrons notamment que l'ASD offre des meilleures performances que les scores classiques de comparaison de fragments sur des tâches concrètes de classification non-supervisée et de fouille structurale. Enfin, grâce à des techniques d'apprentissage automatique, nous mettrons en œuvre la détection de CF à partir de la séquence pour l'identification de protéines de virus avec l'outil VIRALpro développé au cours de cette thèse. / This thesis investigates the local characterization of protein families at both structural and sequential level. We introduce contact fragments (CF) as parts of protein structure that conciliate spatial locality together with sequential neighborhood. We show that the predictability of CF from the sequence is better than that of contiguous fragments and of structurally distant pairs of fragments. In order to structurally compare CF, we introduce ASD, a novel alignment-free dissimilarity measure that respects triangular inequality while being tolerant to sequence shifts and indels. We show that ASD outperforms classical scores for fragment comparison on practical experiments such that unsupervised classification and structural mining. Ultimately, by integrating the identification of CF from the sequence into a statistical machine learning framework, we developed VIRALpro, a tool that enables the detection of sequences of viral structural proteins.

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X-ray crystallography is one of the most powerful tools for the elucidation of the structure of biological macromolecules such as proteins and viruses. Crystallographic techniques are extensively used for investigations on protein structure, ligand-binding, mechanisms of enzyme catalyzed reactions, protein-protein interactions, role of metal ions in protein structure and function, structure of multi-enzyme complexes and viruses, protein dynamics and for a myriad other problems in structural biology. Crystallographic studies are essential for understanding the intricate details of the mechanism of action of enzymes at molecular level. Understanding the subtle differences between the pathogenic enzymes and host enzymes is necessary for the design of inhibitor molecules that specifically inhibit parasite enzymes. The current thesis deals with the application of biochemical and crystallographic techniques for understanding the structure and function of proteins from two pathogenic organisms – a plant virus Physalis Mottle Virus (PhMV), and a pathogenic bacterium, Salmonella typhimurium and also stress induced proteins from Oryza sativa. The thesis has been divided into seven chapters, with the first four chapters describing the work carried out on PhMV, while the rest of the chapters deal with the studies on stress response proteins from Oryza sativa and Salmonella typhimurium. The first part of the thesis deals with studies on viral capsids. Viruses are obligate parasites that have proteinaceous capsids enclosing the genetic material, which, in the case of small plant viruses, is invariably ss-RNA. X-ray diffraction studies on single crystals of viruses enable visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, flexibility of the N-and C-terminal segments and their probable importance in viral assembly, role of RNA in capsid assembly, nucleic acid (RNA)-protein interactions, the capsid structure and mechanism of assembly and disassembly. The present thesis deals with the capsid structure and analysis of the coat protein (CP) recombinant mutants of PhMV. Virus assembly, one of the important steps in the life cycle of a virus, involves specific interactions between the structural protein and cognate viral genome. This is a complex process that requires precise protein-protein and protein nucleic acid interactions. In fact, most of the biological functional units such as ribosomes and proteosomes also require highly co-ordinated macromolecular interactions for their functional expression. Viruses being simple in their architecture, serve as excellent model systems to understand mechanism of macromolecular assembly and provide necessary information for the development of antiviral therapeutics, especially in animal viruses. PhMV is a plant virus infecting several members of Solanaceae family. It belongs to the tymoviridae group of single stranded RNA viruses. Its genome is encapsidated in a shell comprising of 180 (architecture based on T = 3 icosahedral lattice) chemically identical coat protein (CP) subunits (~ 20,000Da) arranged with icosahedral symmetry. In an earlier phase of work, PhMV purified from infected plant leaves was crystallized in the space group R3 (a = 294.56 Å,  = 59.86). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. Using this data of severely limited quality and poor completion (40%), the structure PhMV was determined by molecular replacement using the related turnip yellow mosaic virus (TYMV) structure as the phasing model. There was therefore a need to re-determine and improve the structure, which could be useful for understanding the earlier detailed studies on its biophysical properties. As a continuation of these studies, the present investigations were conceived with the goal of determining the natural top and bottom component capsid structures of PhMV. Investigations were also carried out to examine the possibility of enhancing the diffraction quality of PhMV crystals. The thesis begins with a review of the current literature on the available crystal structures of viruses and their implications for capsid assembly (chapter I). All experimental and computational methods used during the course of investigations are described in chapter II, as most of these are applicable to all the structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. Chapter III describes structural studies on top and bottom components of PhMV. Purified tymoviruses including PhMV are found to contain two classes of particles that sediment at different velocities through sucrose gradients and are called the top (sedimentation coefficient 54 Svedberg units(S)) and the bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA (5%) while the bottom component particles contain the full length genomic RNA. Only the bottom component is infectious. The top and bottom components were separately crystallized in P1 and R3 space groups, respectively. It is of interest to note that crystals of the bottom component obtained earlier belonged to R3 space group while recombinant capsids that lack of full length RNA as in natural top component crystallized in the P1 space group. A polyalanine model of the homologous TYMV was used as the phasing model to determine the structures of these particles by molecular replacement using the program AMoRe. The refinement of top and bottom component capsid structures were carried out using CNS version 1.1 and the polypeptide models were built into the final electron-density map using the interactive graphics program O. The quality of the map was sufficient for building the model and unambiguous positioning of the side chains. There is a significant difference in the radius of the top and bottom component capsids, the top component being 5 Å larger in radius. Thus, RNA makes the capsid more compact, even though RNA is not a pre-requisite for capsid assembly. Partially ordered RNA was observed in the bottom component. The refined models could form the basis for understanding the architecture, protein-protein interactions, protein-nucleic acid interactions, stability and assembly of PhMV. Chapter IV provides a detailed description of the mutations carried out on PhMV coat protein towards enhancing the diffraction quality of crystals. The gene coding for PhMV coat protein (PhMVCP) and several of its deletion and substitution mutants were originally cloned in pRSETC and pET-21 vectors by Mira Sastri and Uma Shankar in Prof. Savithri’s laboratory at the Department of Biochemistry. It was observed that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble into empty shells resembling the natural top component. None of these deletion mutants crystallized in forms that diffracted to high resolution. Based on the intersubunit contacts observed, three more site-specific mutants were designed. These three mutants were expressed in BL21 (DE3), purified and crystallized. Even these mutant crystals did not diffract to high resolution. The polypeptide fold of PhMV coat protein therefore was carefully examined for probable reasons. It was found that PhMV subunit has three major cavities. Three cavities are likely to increase the flexibility of protein subunits, which in turn may result in crystals of poor quality. Mutations V52W, S158Q and A160L were shown to fill up these cavities and with the view of obtaining better crystals. These site specific mutations were carried out the mutant proteins were purified. It was shown that the recombinant capsids are stable and possess T=3 architecture. Two mutants were crystallized and a data set for V52W extending to 6.0 Å resolution could be collected. Due to the limited resolution, further work was not pursued. It is plausible that the triple mutant will diffract to higher resolution. The second part of the thesis deals with stress response proteins from Oryza sativa and Salmonella typhimurium. It is known that viral infection and abiotic and biotic stresses induce a network of proteins in plants. Chapter V presents a review of the current literature on stress proteins, focusing mainly on Oryza sativa and S. typhimurium stress response proteins. Chapter VI describes the over expression of stress proteins SAP1 and SAP2 from rice. These stress related proteins confer tolerance to cold, dehydration and salt stress in rice. These proteins have been cloned in the expression vector pEt-28(a) and expressed in E. coli strain BL21 CodonPlus(DE3)RIL. The proteins were purified and crystallization trials were made. However, there were no hits. In an attempt to get crystals, nine deletion constructs of SAP1 were designed eliminating potentially disordered and unfolded regions based on a bioinformatics analysis. Crystallization trails are being carried out on three of the constructs. Structural studies on a universal stress protein from Salmonella typhimurium, which shares homology with the rice universal stress proteins, was initiated. Apart from this, several other stress related proteins of Salmonella typhimurium have also been selected for structural and functional studies. These include YdaA, YbdQ, Yic, Ynaf, Yec, Spy and Usb. All these were cloned and expressed in E. coli. Out of seven proteins, Ynaf, YdaA and YbdQ were found in the soluble fraction and were expressed in quantities suitable for structural studies. I could crystallize YdaA and Ynaf. X-ray diffraction data to resolutions of 3.6 Å and 2.3 Å were collected on crystals of YdaA and YnaF, respectively. A tentative structure of YnaF has been obtained. Further attempts to determine these structures are in progress. Biophysical, Biochemical functional characterization of YdaA and YnaF proteins are described. Structural studies on mannose-6-phosphate isomerase, an enzyme related to stress regulatory proteins from S. typhimurium are dealt with in Chapter VII. Mannose 6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The structure could be solved in its apo and holo forms (with two different metal atoms, Y3+ and Zn2+), and complexed with the cyclic form of the substrate fructose 6-phosphate (F6P) and Zn2+. Isomerization involves acid/base catalysis with proton transfer between C1 and C2 atoms of the substrate. Lys 132, His 131, His 99 and Asp 270 are close to the substrate and are likely to be the residues involved in proton transfer. Interactions observed at the active site suggest that the ring opening step is catalyzed by His 99 and Asp 270. An active site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. The metal ion is not close to the substrate atoms involved in proton transfer. Binding of the metal induces structural order in the loop consisting of residues 50-54. Hence, the metal atom does not appear to play a direct role in catalysis, but is probably important for maintaining the architecture of the active site. Based on these structures and earlier biochemical work, a probable isomerization mechanism has been proposed. The thesis concludes with a brief discussion on the future prospects of the work. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis:

Chapter 1: Role of JEV NS1 in protective immunity and in immunopathology. Previous studies from our laboratory revealed T cell immunodominance of non structural proteins NS3 and NS1 during natural JEV infections in humans where as the structural protein E, which is a good target for neutralizing antibody response is a poor inducer of T cells. Flavivirus NS1 is also known to induce humoral immune response. Several studies in different flaviviruses have indicated a role for NS1-specific immune responses in protection against flaviviruses. Paradoxically, studies also pointed to the contribution of NS1 in pathology and immune modulation. We screened serum samples from 72 convalescent JE patients for the presence of anti-NS1 antibodies by ELISA and radioimmunoprecipitation and found NS1 reactivity in 45 samples. These antibodies to NS1 are capable of inducing complement mediated cytolysis of cells expressing NS1 on the surface. Additionally, we demonstrated twenty two fold reduction in the infectious virus produced at 48h in SW-13 cells in the presence of human complement and NS1 antiserum compared to control serum, suggesting that complement mediated cytolytic activity of anti NS1 antibody helps the host in controlling the virus propagation. Chapter 2: Comparison of immune responses to JEV structural proteins Capsid and Envelope in human volunteers vaccinated with inactivated JE vaccine and naturally exposed to live JEV. We compared the CMI responses to structural proteins E and C in human volunteers vaccinated with commercially available killed JE vaccine and in humans naturally exposed to live JEV. The results revealed that structural proteins E and C are inherently poor inducers of T cells even in killed vaccine preparation, where there is no competition from immunodominant non structural proteins. Therefore inclusion of nonstructural proteins NS1 and NS3 along with neutralizing antibody inducing envelope should improve memory and efficacy of a JE vaccine. Chapter 3: Construction and testing in the mouse model of experimental recombinant poxvirus vaccines expressing prM, E, NS1, and NS3 of JEV. Guided by the information on immune responses to JEV in the JE endemic human cohort and volunteers vaccinated with killed JE vaccine, we designed experimental vaccines as recombinant vaccinia viruses expressing NS1, NS3, prM, and E proteins of JEV (vNS1NS3prME) or NS1, NS3, prM, and C-terminally truncated E (vNS1NS3prMΔE) and studied the immune responses elicited by these vaccines in mice. Our data showed that a recombinant vaccinia virus expressing prM, ΔE, NS1, and NS3 of JEV is superior to killed JE vaccine in eliciting long lived neutralizing antibodies as well as NS1 and NS3-specific cytotoxic T lymphocytes (CTL) in addition to NS1-specific cytolytic antibodies, resulting in long lasting and enhanced protection from lethal JEV infection in mice. Our results thus identified all B and T cell antigens whose inclusion in a live-vectored vaccine would provide a vaccine with far superior efficacy over the inactivated JE vaccine.

L’hépatite B est une maladie du foie qui pose un problème majeur de santé publique. Il n’existe à ce jour aucun traitement permettant de guérir complètement de l’infection, et de nouvelles thérapies ont besoin d’être développées. Étant donné son rôle clé dans le cycle de vie du virus de l’hépatite B (VHB), la protéine core qui forme la capside virale est aujourd’hui l’une des cibles avec le plus grand potentiel thérapeutique. Nos recherches sont focalisées sur la caractérisation des capsides du VHB dans différents états conformationnels en utilisant des techniques de biochimie et de RMN du solide, afin de révéler leur conformation précise sous différentes conditions, incluant l’interaction des capsides avec des antiviraux, et la relation entre la conformation de la capside et la maturation du virus. Un système d’expression bactérienne ainsi qu’un système acellulaire de synthèse de protéine à base de germes de blé ont été établis au laboratoire pour produire les capsides, et des protocoles pour désassembler puis réassembler les capsides en présence de différents types d’ARN ont été implémentés. Des échantillons de capsides formées dans E. coli et réassemblées in vitro ont été analysés par RMN. Les différentes formes de capsides observées incluent les protéines tronquées Cp140 et Cp149, la protéine entière Cp183, phosphorylée P-Cp183, et enfin des mutants. Dans un premier temps, nous avons préparé des échantillons pour l’attribution séquentielle de la protéine core par RMN du solide. L’utilisation de la détection carbone en RMN requiert plusieurs dizaines de milligrammes d’échantillon, qui ont pu être produits en utilisant l’expressions bactérienne en milieu minimum contenant des isotopes marqués. Les attributions séquentielles ont été réalisées sur la protéine tronquée Cp149, qui donne des spectres très similaires à Cp183. Cet échantillon a également été utilisé pour identifier les différences conformationnelles entre les 4 monomères de la capside, qui sont provoquées par la symétrie icosaédrale T=4. Ensuite, l’objet principal de cette thèse a été l’investigation et la comparaison d’une large variété de capsides, dans leur forme autoassemblée dans les bactéries E. coli, ainsi que dans leur forme réassemblée. Pour le réassemblage de la protéine entière, qui requiert la présence d’acides nucléiques, nous avons testé différents types d’ARN y compris l’ARN viral prégénomique. Nous avons étudié différentes symétries (T=3 et T=4), ainsi que les états d’oxydation de la capside, et comparé les différences de conformation grâce aux perturbations de déplacements chimiques observées dans les spectres RMN. Nous avons pu identifier les acides aminés impliqués dans les changements conformationnels majeurs entre les différentes préparations. La RMN du solide en détection proton à 100 kHz a récemment émergé comme un outils important pour l’analyse de protéines produites en quantités moindres. Nous avons appliqué cette stratégie à l’analyse des capsides de Cp149 afin d’obtenir l’attribution des protons amides. La détection proton par RMN du solide peut être combinée avec succès à la synthèse des protéines en système acellulaire, qui donne de faibles rendements par rapport aux cultures en bactéries. Cette approche est particulièrement intéressante pour analyser la modulation de l’assemblage des capsides induite par la présence de drogues. Bien que nous ayons commencé à étudier l’impact de modulateurs d’assemblage par RMN en détection carbone sur des capsides formées dans E. coli et réassemblées (données préliminaires non montrées dans ce manuscrit), la détection proton ouvre la voie vers l’analyse de l’impact de ces modulateurs sur l’assemblage des protéines core directement à la sortie du ribosome / Hepatitis B is a widely spread liver disease which causes a heavy burden for human health, with 257 millions of people affected by chronic infection and about 780,000 deaths per year. Yet, infected patients can not be completely cured by current treatments using notably nucleos(t)ide analogues and interferons. In order to achieve the goal of the World Health Assembly (WHA), who wishes to eliminate hepatitis B by 2030, new therapies need to be developed. Given its critical role for the Hepatitis B virus (HBV) life cycle, the core protein (Cp) is today one of the antiviral targets with the highest potential. Our research focuses on the characterization of HBV capsids in different conformational states using biochemistry and solid-state NMR, aiming at revealing their precise conformation under different conditions, including the interaction of capsids with antivirals, and the correlation between capsid conformation and viral maturation. For sample preparation, both a bacterial expression system and a wheat germ cell-free protein synthesis system have been established in the laboratory to produce HBV capsids, and protocols to disassemble and reassemble capsids with different nucleic acids have been implemented. Both capsids preformed in E. coli and capsids reassembled in vitro were addressed to NMR studies. Different capsids forms include the truncated versions Cp140 and Cp149, the full length protein Cp183, the phosphorylated P-Cp183 and mutant forms. First, we have prepared samples for the sequential assignment of the protein using solid-state NMR. The use of carbon-13 detection asks for several tens of milligrams of sample, which were produced using labeled isotopes and bacterial expression in minimal media. Sequential assignments were performed using the truncated capsid Cp149, which showed highly similar spectra to Cp183. This sample was also used to identify conformational differences between the four different monomers in the capsid, which are due to the T=4 icosahedral symmetry. Then, the main body of the thesis is the investigation and comparison of a variety of different capsid forms, including Cp183, P-Cp183, Cp149, Cp140, another truncated form resulting in mainly T=3 icosahedral assemblies, and Cp140 C61A and Cp183 F97L mutants. We investigated all samples in both the E. coli-produced and reassembled forms, which needs for the full-length protein the presence of nucleic acids, of which we tested several, including the viral pregenomic RNA. We investigated different symmetries, as well as oxidation states of the capsid, and compared the differences via chemical shift perturbations observed in NMR spectra. We reported in a site-specific manner the major conformational changes observed between the different preparations. Proton-detected solid-state NMR at 100 kHz has recently emerged as a tool for analyzing proteins with the need of less sample amount. We have applied this strategy to the analysis of the Cp149 capsids, in order to obtain sequential assignments of the amide proton resonances. For this, deuteration of the protein in bacteria was used as well, needing adaptation of sample preparation protocols. Proton detection can be successfully combined with cell-free protein synthesis, which gives low yields compared to bacterial expression. This approach is of potential interest to analyze capsid assembly modulation induced by the presence of drugs. While we have started in the framework of this thesis to analyze the capsid in presence of different capsid assembly modulators by carbon-13 detected NMR on E. coli and reassembled capsids (preliminary results not reported here), proton detection opens the way to an analysis of the impact of capsid modulation directly on the exit of the core proteins from the ribosome, on assembly. We showed that cell-free expression combined with proton-detection solid-state NMR can be used to analyze capsid chemical shifts, and thus in future work the conformational modulations

Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...

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