The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry

<p>Abstract</p> <p>Background</p> <p>Three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for <it>Plasmodium </it>infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM).</p> <p>Results</p> <p>In our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels.</p> <p>Conclusion</p> <p>In contrast to <it>Plasmodium </it>infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.</p

Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment

The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry

Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor

CARACTERISATION DES DOMAINES TRANSMEMBRANAIRES DES GLYCOPROTEINES E1 ET E2 DU VIRUS DE L'HEPATITE C

DANS CE TRAVAIL DE THESE, NOUS NOUS SOMMES INTERESSES AUX ETAPES PRECOCES DE LA FORMATION DE L'ENVELOPPE VIRALE DU VIRUS DE L'HEPATITE C (VHC). LE GENOME DU VHC CODE DEUX GLYCOPROTEINES D'ENVELOPPE APPELEES E1 ET E2. CES GLYCOPROTEINES POSSEDENT UN LARGE ECTODOMAINE N-TERMINAL HAUTEMENT GLYCOSYLE ET SONT ANCREES DANS LES MEMBRANES PAR LEUR DOMAINE TRANSMEMBRANAIRE (TM) C-TERMINAL. CES DEUX GLYCOPROTEINES INTERAGISSENT DE MANIERE NON COVALENTE POUR FORMER UN HETERODIMERE E1E2 QUI EST SUPPOSE ETRE LE COMPOSANT PROTEIQUE MAJEUR DE L'ENVELOPPE DU VHC. LORSQU'IL EST EXPRIME PAR DES VECTEURS D'EXPRESSION HETEROLOGUE, L'HETERODIMERE E1E2 EST RETENU AU NIVEAU DU RETICULUM ENDOPLASMIQUE (RE). DANS NOTRE TRAVAIL, NOUS AVONS DANS UN PREMIER TEMPS CHERCHE A IDENTIFIER LES DETERMINANTS IMPLIQUES DANS CETTE LOCALISATION SUBCELLULAIRE DU COMPLEXE E1E2. PAR LE BIAIS DE CONSTRUCTIONS DE PROTEINES CHIMERIQUES ENTRE LES PROTEINES D'ENVELOPPE DU VHC ET DES PROTEINES NORMALEMENT EXPORTEES A LA MEMBRANE PLASMIQUE (CD4 ET/OU CD8), NOUS AVONS MONTRE QUE LES DOMAINES TMS DE E1 ET E2 SONT DES SIGNAUX DE RETENTION STRICTE DANS LE RE. PAR UNE APPROCHE DE MUTAGENESE DIRIGEE, NOUS AVONS ENSUITE MONTRE QUE LA PRESENCE D'UN OU DE DEUX RESIDUS CHARGES, LOCALISES AU MILIEU DES DOMAINES TMS DE E1 ET E2, JOUENT UN ROLE ESSENTIEL DANS LA LOCALISATION SUBCELLULAIRE DE CES PROTEINES. DE PLUS, NOUS AVONS CONSTATE QUE CES RESIDUS CHARGES SONT EGALEMENT IMPORTANTS POUR L'ASSEMBLAGE DU COMPLEXE E1E2 AINSI QUE POUR LA FONCTION SEQUENCE SIGNAL SITUEE DANS LA MOITIE C-TERMINALE DE CES DOMAINES. ENFIN, NOUS AVONS DETERMINE LA TOPOLOGIE DE CES DOMAINES TMS AVANT ET APRES CLIVAGE DE LA POLYPROTEINE DU VHC ET NOUS AVONS MIS EN EVIDENCE UNE DYNAMIQUE STRUCTURALE DANS LES ETAPES INITIALES DE LA BIOGENESE DES PROTEINES D'ENVELOPPE DU VHC. L'ENSEMBLE DE CE TRAVAIL NOUS PERMET DE PROPOSER UN MODELE RELIANT LA MULTIFONCTIONNALITE DES DOMAINES TMS DES PROTEINES D'ENVELOPPE DU VHC A LEUR DYNAMIQUE STRUCTURALE.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Le virus de l’hépatite E: Un virus méconnu qui se dévoile

International audienceThe first cause of acute hepatitis in the world is due to the hepatitis E virus (HEV). This infection has long been considered as a problem only affecting developing countries. However, since the identification of zoonotic forms at the end of the last century, it has become clear that this infection also affects industrialized countries. The recent renewed interest in HEV has revealed some particularities in this virus. Indeed, although considered as a non-enveloped virus, the HEV viral particle is surrounded by a lipid membrane in the bloodstream. In addition, HEV secretes abundantly into the bloodstream non-infectious forms of its capsid protein that could serve as an immunological bait. This review summarizes recent advances on this virus for which the number of diagnosed cases increases every year.La première cause d'hépatite aiguë dans le monde est due au virus de l'hépatite E (HEV). Cette infection virale a longtemps été considérée comme un problème touchant les pays en déve-loppement. Cependant, depuis l'identification de formes zoonotiques à la fin du siècle dernier, il est apparu que cette infection touche éga-lement les pays industrialisés. Le récent regain d'intérêt pour le HEV révèle des particularités de ce virus. Bien que considéré comme un virus non enveloppé, la particule virale du HEV est entou-rée d'une membrane lipidique dans la circulation sanguine où le virus produit en abondance des formes de sa protéine de capside qui ne sont pas associées à du matériel infectieux et qui pourraient servir de leurre immunologique. Cette revue résume les avancées récentes sur ce virus, dont le nombre de cas d'infection diagnostiqués augmente chaque année

Charged Residues in the Transmembrane Domains of Hepatitis C Virus Glycoproteins Play a Major Role in the Processing, Subcellular Localization, and Assembly of These Envelope Proteins

For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope