No description available.

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No description available.

Lassa fever virus is one of the most dangerous viruses of arenaviridae family, causing more than 500,000 infections per year in Africa. The fatality rate for hospitalized patients is as high as 20%. Due to the high fatality and lack of efficient licensed drugs and vaccines to treat and prevent, Lassa fever virus is classified as a Category A priority pathogen and biosafety level-4 agent by the Centers for Disease Control and Prevention of the USA. Cases were also found in the Americas and European countries, highlighting its potency to be a bioterrorism weapon. Like other areanaviruses, Lassa virus has developed a unique interferon suppression mechanism to evade from the host immune system, in which Lassa nucleoprotein plays the key role. To understand the LASV nucleoprotein functions, we tried to determine the first arenaviral nucleoprotein structure, LASV nucleoprotein. The LASV nucleoprotein (NP) was overexpressed and purified. The NP protein was crystallized and the structure was determined to 1.80 Å resolution. The crystals belong to space group P3, with the unit cell parameters a = b = 177.16 Å, c = 56.49 Å, α= β= 90° and γ= 120°. The LASV NP structure contains two domains, which are not similar to any reported viral nucleoprotein structures. The N-terminal domain has a novel structure with a cavity, which we proposed for cap binding, and the C-terminus is a 3'-5' ribonuclease, which is responsible for suppressing interferon production. To characterize the possible interaction between NP and other arenaviral protein, we also overexpressed and purified LASV Z. Interestingly, both NP and Z proteins have two forms and the purified NP protein and monomeric Z protein bind RNA. It is surprising that only the oligomeric Z protein interacts with NP protein but the monomeric Z protein does not as determined by Isothermal Titration Calorimetry (ITC). Our studies have reported the first arenaviral nucleoprotein structure, revealed the novel mechanism for the cap binding and immune suppression, which set up a platform for the development of novel drugs and vaccines to treat deadly arenaviral infections.

Human respiratory syncytial virus (HRSV) is a leading cause of virus-induced paediatric respiratory disease. HRSV can cause severe lower respiratory tract diseases in young children and upper respiratory tract diseases in all ages. HRSV infection results in large economical and healthcare burdens every year, however, there is no approved vaccine and the antiviral therapies are generally costly and less effective due to the high mutant rate of this RNA virus and the imbalanced host immune responses in response to HRSV infection. The virus-host interactions of HRSV have not been well understood so far, which therefore limited the knowledge of HRSV pathogenesis and the development of vaccinations and antiviral drugs. This study focused on investigations of the interactions between HRSV and host NF-KB activation and host cell cycle manipulation, as well as the interactions between HRSV nonstructural proteins and host cellular proteins. In conclusion, different NF-KB activation characteristics have been found between HRSV subgroups A and B, suggesting the implication with the different pathology of HRSV subgroup A and B. HRSV infection resulted in cell cycle arrest at GO/G1 phase with different mechanism in continuous and primary cell cultures, which benefited progeny virus production. HRSV NS1 protein was found to act as a role in HRSV induced cell cycle arrest, and a potential inhibitor of RNA polymerase". All of these findings provided a better understanding of HRSV virus-host interactions and HRSV pathogenesis.

No description available.

This thesis examines the evolutionary biology of the RNA viruses, a diverse group of pathogens that cause significant diseases. The focus of this work is the relationship between the processes driving the evolution of virus populations within individual hosts and at the epidemic level. First, Chapter One reviews the basic biology of RNA viruses, the current state of knowledge in relevant topics of evolutionary virology, and the principles that underlie the most commonly used methods in this thesis. In Chapter Two, I develop and test a novel framework to estimate the significance of phylogeny-trait association in viral phylogenies. The method incorporates phylogenetic uncertainty through the use of posterior sets of trees (PST) produced in Bayesian MCMC analyses. In Chapter Three, I conduct a comprehensive analysis of the substitution rate of hepatitis C virus (HCV) in within- and between-host data sets using a relaxed molecular clock. I find that within-host substitution rates are more rapid than previously appreciated, that heterotachy is rife in within-host data sets, and that selection is likely to be a primary driver. In Chapter Four I apply the techniques developed in Chapter Two to successfully detect compartmentalization between peripheral blood and cervical tissues in a large data set of human immunodeficiency virus (HIV) patients. I propose that compartmentalization in the cervix is maintained by selection. I extend the framework developed in Chapter Two in Chapter Five and explore the Type II error of the statistics used. In Chapter Six I review the findings of this thesis and conclude with a general discussion of the relationship between within- and among-host evolution in viruses, and some of the limitations of current techniques.

On assiste actuellement à l'émergence et la ré-émergence mondiale d'arboviroses comme le chikungunya, la dengue ou la fièvre de la vallée du Rift. Ces maladies, responsables d'environ 30 000 décès par an, sont dues à des virus principalement transmis à l'homme par des moustiques vecteurs. En l'absence de vaccins efficaces et face aux limites de l'utilisation d'insecticides, nocifs pour les écosystèmes et entrainant des résistances chez les vecteurs, il est nécessaire de développer des moyens alternatifs de lutte. La découverte récente du potentiel antiviral de certaines bactéries symbiotiques du moustique, comme le genre Wolbachia, représente un outil de lutte biologique prometteur face aux arboviroses. Ce projet de thèse porte sur la relation tripartite moustique-bactéries endosymbiotiques-arbovirus, en prenant pour modèle le moustique-tigre, Aedes albopictus. Cette espèce originaire d'Asie envahit progressivement l'Europe. Elle transmet notamment le virus de la dengue et du chikungunya, et est naturellement infectée par Wolbachia. Les résultats obtenus ont permis d'observer un phénotype antiviral chez les moustiques infectés par Wolbachia, contrairement aux moustiques aposymbiotiques. L'utilisation d'une méthode de transcriptomique haut-débit (RNAseq) a permis de déterminer certains mécanismes cellulaires et moléculaires majeurs du moustique spécifiquement impliqués dans l'interaction avec les arbovirus, Wolbachia, et les deux partenaires simultanément. Le développement d'une lignée cellulaire d'Ae. albopictus stablement infectée par Wolbachia a permis de mettre en évidence le rôle central de l'autophagie dans l'interaction Wolbachia-arbovirus chez Ae. Albopictus / Arthropod-borne virus (arbovirus) are important cause of human diseases worldwide, leading to nearly 30.000 deaths every year. Many arboviruses like dengue virus (DENV), chikungunya virus (CHIKV) or Rift valley Fever virus (RVFV) are transmitted by mosquitoes, and global changes like climate warming or international trade increase vectors geographic range, thus facilitating the emergence of arbovirosis. Very few vaccines are currently available, and the use of insecticides remains the only way to prevent arbovirosis but cause adverse effects on ecosystems, and lead to resistance phenotypes in vector populations. Recent work showed that mosquito bacterial flora, especially bacteria from the genus Wolbachia, can modulate viral infection, a phenotype called microbial interference. These results provide a promising tool to limit transmission of arboviruses, but little is known about mosquito-Wolbachia-arbovirus interaction especially at the cellular level. We characterized for the first time this multipartite interaction in Aedes albopictus, an important mosquito vector of DENV and CHIKV, which is naturally infected by Wolbachia. Results showed an antiviral phenotype in Wolbachia-infected mosquitoes, compared to aposymbiotic insects. We used RNAseq to decipher the major mosquito pathways implemented during mono-infection by virus, bacteria or during bi-infection. Moreover, we developed an Ae. albopictus cell line stably infected by Wolbachia to go further in mechanical aspects, and showed that autophagy is a major pathway involved in Wolbachia-arbovirus interaction in Ae. albopictus

Les Rotavirus appartiennent à la famille des Reoviridae, famille du groupe III des virus à ARN double brin. Identifiés en 1973 par Ruth Bishop, ces virus non enveloppés sont la première cause de diarrhée aiguë sévère du jeune enfant dans le monde. La capside virale icosaédrique est constituée de 3 couches protéiques de structure : la couche externe formée par la glycoprotéine VP7 d’où émergent les spicules de protéine VP4, la couche intermédiaire constituée par la protéine VP6 représentant près de 50 % du poids du virus et enfin, la couche interne appelée core, résultant de l’assemblage des protéines VP2, d’où émergent vers l’intérieur les protéines VP1 et VP3. Cette capside renferme un génome divisé en 11 segments d’ARN bicaténaires. A ces 6 protéines structurales s’ajoutent les protéines non structurales qui interviennent lors de la réplication du virus. Les deux protéines structurales, VP4 et VP7 sont essentielles pour la fixation de la particule triple couche (TLP) aux membranes des cellules hôtes, par interaction aux récepteurs intégrines, elle sont également impliqués dans la déstabilisation des membranes endosomales, indispensable à la libération de la particule double couche (DLP) infectieuse dans le cytoplasme. Actuellement, contrairement au mécanisme d’action de la protéine VP5*, celui de la glycoprotéine VP7 est inconnu. L’objectif de cette thèse, a été de comprendre le mécanisme moléculaire de déstabilisation des membranes par les peptides dérivés de VP7. Dans un premier temps nous avons montré, par des études in silico, l’existence d’un domaine prédit en hélice membranaire bordé de résidus arginine et lysine hautement conservés, situé à l’extrémité C-terminale de la glycoprotéine VP7. Ces résultats ont conduit à la synthèse de quatre peptides avec lesquels des tests de perméabilisation de membranes modèles de larges vésicules unilamellaires (LUVs) ont été menés. Ceux-ci ont permis d’identifier le domaine minimum le plus actif, VP723, parmi les peptides sélectionnés. Dans un second temps nous avons déterminé la structure de ces peptides par RMN, dans des conditions mimant l’environnement hydrophobe de la membrane. Le peptide minimal VP723 s’organise en hélice α-amphipathique, structure souvent impliquée dans la déstabilisation des membranes cellulaires. La comparaison de sa structure obtenue par RMN à celle du domaine correspondant dans la structure cristallographique de la protéine native montre le réarrangement conformationnel de ce segment après maturation par la trypsine. Ces résultats ont été confirmés par deux mutants de synthèse, dont l’un est inactif pour la perméabilisation des membranes modèles. Ces travaux ont été complétés par des expériences de Résonance Plasmonique aux Ondes guidée (PWR). Des études par RMN du solide sont en cours afin de déterminer l’orientation du peptide dans les membranes modèles. En conclusion, nos résultats mettent en évidence l’importance du domaine C-terminal VP723 de la protéine VP7 dans la déstabilisation des membranes, permettant d’assurer la translocation de la particule virale infectieuse (DLP) de l’endosome vers le cytoplasme. Un modèle du mécanisme d’entrée du virus, médié par les peptides dérivés de la maturation par la trypsine de la glycoprotéine VP7 est proposé. / Rotaviruses belong to the Reoviridae family, belonging to the group III of dsRNA viruses. Identified in 1973 by Ruth Bishop, these non-enveloped viruses are the leading cause of severe diarrhea in young children worldwide. The icosahedral capsid is composed of three structural protein layers: the outer one, formed by the glycoprotein VP7, emerges spicules protein VP4, the intermediate one consists of VP6 protein representing nearly 50% of the weight of the virus and finally, the inner one called core, results from the assembly of proteins VP2, emerges towards the inside of proteins VP1 and VP3. The capsid contains a genome divided into 11 segments of dsRNA. To these six structural proteins are added nonstructural proteins involved in virus replication. The two structural proteins, VP4 and VP7, are involved in the interaction of the triple layer particle (TLP) to integrin receptors, necessary for the release of the infectious double layer particle (DLP) into the cytoplasm following the permeabilization of the membrane of the endosome compartments. Currently, unlike the mechanism of action of the protein VP5*, the glycoprotein VP7 remains unknown. The objective of this work was to understand the molecular mechanism involved in the destabilization of membranes by peptides derived from VP7. In a first step, we have shown, by in silico studies, the existence of a helical trans-membrane domain predicted containing a highly conserved arginine and lysine residues, located at the C-terminus of the VP7 glycoprotein. These results led to the synthesis of four peptides with which permeabilizing tests of model membranes were conducted. We have identified the minimum of the most active domain, named VP723, among the selected peptides. In a second step, we determined the structure of these peptides by NMR under conditions mimicking the hydrophobic environment of the membrane. The VP723 peptide is organized like an α-helical amphipathic structure often involved in the destabilization of cell membranes. The comparison of the structure obtained by NMR to that of the corresponding domain in the crystallographic structure of the native protein shows a conformational rearrangement of the segment after trypsin maturation. These results were confirmed by two synthetic mutants, one of which is inactive for the permeabilization of model membranes. These studies were complemented by experiments Plasmon Resonance guided the Waves (PWR). Studies by solid state NMR are in progress to determine the orientation of the peptide in model of membranes. In conclusion, our results highlight the importance of the C-terminal domain of the VP7 protein, named VP723, in the destabilization of membranes, to ensure the translocation of the infectious viral particle (DLP) from the endosome into the cytoplasm compartments. A mechanism of virus entry mediated by peptides derived from trypsin maturation of the VP7 glycoprotein is proposed in this study.