Caractérisation structurale de glycoprotéines d'enveloppes virales

Viral glycoproteins are responsible for the two major steps in entry into host cells by enveloped viruses: 1) attachment to cellular receptor/s and 2) fusion of the viral and cellular membranes. My thesis concentrated first on the structural analysis of the major envelope glycoprotein E2 of two hepaciviruses: GB virus B (GBV-B) and hepatitis C virus (HCV). Crystallization of the GBV-B E2 ectodomain remained unsuccessful, but the characterization of truncated versions of E2 suggested an important role of its C-terminal moiety in receptor binding. In parallel, I co-crystallized a synthetic peptide mimicking HCV E2 with an antibody fragment directed against the major receptor-binding loop of E2 that is targeted by broadly neutralizing antibodies. The structure unexpectedly revealed an α-helical peptide conformation, which is in stark contrast to the extended conformation of this region observed in the structure of an E2 core fragment. Together with further biochemical evidence this suggests an unanticipated structural flexibility within this region in the context of the soluble E2 ectodomain. Secondly, I focused on the structural analysis of the baculovirus glycoprotein F. I determined the crystal structure of the post-fusion trimer of a trypsin-truncated F fragment. This structure confirmed previous predictions that baculovirus F protein adopts a class I fusion protein fold and is homologous to the paramyxovirus F protein. Baculovirus F is therefore the first class I fusion protein encoded by a DNA virus. My results support the hypothesis that F proteins may have a common ancestor and imply interesting evolutionary links between DNA and RNA viruses and their hosts.Les glycoprotéines virales sont impliquées dans les deux principales étapes d’entrée des virus enveloppés dans leurs cellules hôtes : l’attachement des virus aux récepteurs cellulaires et la fusion des membranes virale et cellulaire. Je me suis d’abord attachée à l’étude structurale de la principale glycoprotéine, E2, de deux hépacivirus : la forme B du virus GB (GBV-B) et le virus de l’hépatite C (HCV). Mes tentatives de cristallisation de l’ectodomaine de la protéine E2 du GBV-B sont restées vaines, mais l’analyse des propriétés de ses fragments a suggéré un rôle de son extrémité C-terminale dans la liaison à son récepteur. En parallèle, j’ai co-cristallisé un peptide synthétique correspondant à la principale boucle de liaison de E2 à son récepteur, avec un fragment d’anticorps dirigé contre cette boucle. Etonnament, le peptide forme une hélice , en nette contradiction avec la conformation étendue adoptée dans un fragment du cœur de E2. Associé à des données biochimiques, cela suggère une flexibilité inattendue de cette région de l’ectodomaine d’E2. Dans un second temps, je me suis intéressée à la glycoprotéine F des baculovirus. J’ai résolu la structure du trimère d’un fragment tryptique de F dans sa conformation post-fusion. Cette structure a validé une prédiction selon laquelle la protéine F était une protéine de fusion de classe I homologue à celle des paramyxovirus. La protéine F des baculovirus est ainsi le premier exemple d’une protéine de fusion de classe I encodée par un virus à ADN. Mes résultats confortent donc l’hypothèse que toutes les protéines F ont un ancêtre commun et suggèrent un lien évolutif intéressant entre les virus à ADN, à ARN et leurs hôtes

Molecular insights into the biased signaling mechanism of the mu-opioid receptor

GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the beta-arrestin pathways through the mu-opioid receptor (mu OR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific mu OR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs, Most strikingly, the biased agonists trigger mu OR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair beta-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.1

Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2

International audienceABSTRACT The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like β-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a β-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design. IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development

Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

International audienceNuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure

Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction

International audienceHepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts