Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81(+) cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis.
Le virus de l'hepatite C (VHC) est un pathogene majeur causant des cancers du foie. Ce n'est que plus de dix ans apres sa decouverte que des systemes experimentaux in vitro ont ete developpe et ont permis de montrer que le tropisme du virus pour les hepatocytes est restreint par l'interaction des glycoproteines d'enveloppe E1E2 avec des recepteurs de surface cellulaire. Les details de cette etape et le mecanisme conduisant a l'internalisation du VHC ont longtemps ete ignores. Mon travail de these c'est focalise sur l'etude de ces stades precoces du cycle viral, des cibles privilegiees pour le developpement d'un vaccin. Nos travaux ont mis en evidence que la grande boucle extracellulaire du corecepteur CD81 est importante pour l'attachement des particules. Cette etape est suivie de l'intervention de molecules recrutees au sein du complexe virus-recepteurs par les domaines transmembranaires et intracellulaires de CD81 afin d'initier l'internalisation des VHCpp. Nous avons egalement decouvert que les claudines, proteines des jonctions serrees, sont des cofacteurs necessaire a l'entree du VHC. Enfin, nous avons demontre que le VHC est endocyte par la voie de la clathrine et necessite un passage par les endosomes precoces mais non tardifs. Une etude detaillee des cinetiques d'entree des VHCpp a montre que la fusion du virus dependante du pH requiere d'autres evenements au cours du trafic intracellulaire. Ces decouvertes ont permis d'etablir que le mecanisme d'entree du VHC est complexe et nouveau. Elles ouvrent de nouvelles perspectives pour developper des strategies therapeutiques ciblant des etapes specifiques de l'infection.
Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.
Le mot finlandais du Sisu est nécessaire pour comprendre le système de défense finlandais. Ce mot, intraduisible en français, est utilisé pour décrire la ténacité et la résilience de la société finlandaise. Or, c’est ce que reflète le modèle finlandais, la volonté d’une population toute entière de défendre son territoire. Cette volonté s’est forgée par l’histoire du pays et son voisinage avec la Russie. Cela explique les choix qui ont été faits en matière de défense comme celui d’une armée de conscription et d’une réserve forte. Cela permet également de comprendre que le modèle de défense finlandais est un modèle global qui met l’ensemble de la société au service de la défense territoriale.
The CD81 tetraspanin was first identified as a hepatitis C virus (HCV) receptor by its ability to bind the soluble ectodomain of envelope glycoprotein E2 (sE2). More recently, it has been suggested that CD81 is necessary but not sufficient for HCV entry into target cells. Here we present further evidence that putative human hepatocyte-specific factors act in concert with CD81 to mediate sE2 binding and HCV pseudoparticle (HCVpp) entry. Moreover, we show that CD81-mediated HCVpp entry entails E2 binding to residues in the large extracellular loop as well as molecular events mediated by the transmembrane and intracellular domains of CD81. The concept that CD81 receptor function progresses in stages is further supported by our finding that anti-CD81 monoclonal antibodies inhibit HCVpp entry by different mechanisms. The half-life of CD81-HCVpp binding was determined to be approximately 17 min, and we propose that binding is followed by CD81 oligomerization, partitioning into cholesterol-rich membrane domains, or other, lateral protein-protein interactions. This results in the formation of a receptor-virus complex that undergoes endocytosis and pH-dependent membrane fusion.
ABSTRACT Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W 30 -GLW 51 -C 54 -C 64 . Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.
Focal adhesion disassembly is regulated by microtubules (MTs) through an unknown mechanism that involves dynamin. To test whether endocytosis may be involved, we interfered with the function of clathrin or its adaptors autosomal recessive hypercholesteremia (ARH) and Dab2 (Disabled-2) and found that both treatments prevented MT-induced focal adhesion disassembly. Surface labeling experiments showed that integrin was endocytosed in an extracellular matrix-, clathrin-, and ARH- and Dab2-dependent manner before entering Rab5 endosomes. Clathrin colocalized with a subset of focal adhesions in an ARH- and Dab2-dependent fashion. Direct imaging showed that clathrin rapidly accumulated on focal adhesions during MT-stimulated disassembly and departed from focal adhesions with integrin upon their disassembly. In migrating cells, depletion of clathrin or Dab2 and ARH inhibited focal adhesion disassembly and decreased the rate of migration. These results show that focal adhesion disassembly occurs through a targeted mechanism involving MTs, clathrin, and specific clathrin adaptors and that direct endocytosis of integrins from focal adhesions mediates their disassembly in migrating cells.
