Biochemical and functional characterisation of virion-associated Hepatitis C virus envelope glycoproteins

par Gabrielle Vieyres

Thèse de doctorat en Virologie

Sous la direction de Jean Dubuisson et de Arvind H. Patel.

Soutenue en 2010

à Lille 2 .

mots clés

• Virus de l'hépatite C
• Hépatite C
• Protéines de l'enveloppe virale
• Glycoprotéines
• Glycoprotéines
• Anticorps antivirus
• Anticorps de l'hépatite

Résumé

Hepatitis C is a major global health burden with unsatisfactory treatments and no vaccine available. It is caused by a small positive-strand RNA enveloped virus, namely hepatitis C virus (HCV). HCV encodes 10 proteins, including three structural proteins, the capsid protein (Core) and two envelope proteins (E1 and E2), the focus of this thesis. My thesis first concentrated on E2 hypervariable region 1 (HVR1). HVR1 constitutes an immunological decoy as it elicits isolate-specific antibodies but continuously escapes their neutralisation by adapting its aminoacid sequence. Using a set of new anti-HVR1 antibodies, we found that the C-terminus of HVR1 carries virus neutralising determinants and defined key residues in their epitope. Furthermore, anti-HVR1 antibodies did not inhibit E2 interaction with CD81 or heparin but still inhibited cell-cultured HCV (HCVcc) infection after virus attachment had occurred, confirming a role for HVR1 in the active steps of entry. In addition, we developed a new method to synchronise HCV infection, based on magnetic adsorption. Briefly, HCV was combined with magnetic beads and pulled down onto the cell surface under a magnetic field. This proved to be both very simple and highly efficient as HCV particles were found to be adsorbed in only two minutes onto the cell surface, more efficiently than after a standard, on-synchronised infection. This new method is amenable to study early steps of virus infection, while optimising the infection efficiency. Lastly, the characteristics of HCV glycoproteins in the HCVcc system were analysed. Importantly, the secreted envelope glycoproteins were mostly associated with infectious virions, despite the low specific infectivity inherent to the HCVcc system. Importantly, while intracellular glycoproteins were competent for interaction with entry factors (glycosaminoglycans and CD81) they were found to undergo drastic changes during virion morphogenesis and secretion. Indeed, some E2 glycans were modified and most importantly, virion-associated glycoproteins formed high molecular mass covalent complexes that retained a functional conformation. This study is the first one to describe envelope glycoproteins incorporated onto infectious HCV particle and uncovers unexpected features in HCV assembly. Altogether, this work permitted to delineate more precisely a neutralising epitope in E2 HVR1 and its function in entry. It also gave further insights into the incorporation of HCV glycoproteins in the virion envelope. Together, these results will contribute significantly to future studies aimed at understanding the processes involved in HCV assembly and entry.

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