Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Abstract

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection. Hepatitis C virus (HCV)3 entry into permissive cells is initiated through its E1 and E2 glycoprotein interactions with at least four cellular co-factors as follows: CD81; scavenger receptor class B, type I (SR-BI); and two tight junction-resident proteins, claudin-1 and occludin (1-6). CD81 belongs to the tetraspanin family and thus contains two extracellular loops, of which the second larger loop (ECL2) specifically binds to the HCV E2 glycoprotein (7, 8). SR-BI, which is normally involved in the regulation of lipoprotein metabolism and cholesterol trafficking (9), plays a role in early steps of HCV entry (10 -12); however, its direct interaction with the virus has not been unambiguously demonstrated. Claudin-1 and occludin also consist of four transmembrane domains but are functionally distinct from tetraspanins. The HCV entry determinants of claudin-1 and occludin have been mapped to the first and the second extracellular loops of these proteins, respectively (4,13,14). Considering that all four HCV entry co-factors are expressed in non-liver tissues (1, 5), the narrow tropism of this virus is surprising. It is thus likely that the relative expression levels of entry co-factors, their spatial organization, and/or interactions with each other render a cell permissive to infection (15). An alternative possibility suggested in a rece...

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Entry and replication of recombinant hepatitis C viruses in cell culture

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Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

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