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

Nishi R Sharma,Guaniri Mateu,Marlene Dreux,Arash Grakoui,François-Loïc Cosset,Gregory B Melikyan

doi:10.1074/jbc.m111.263350

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.

Highlights

Infection by cell-attached and immobilized viruses was sensitive to bafilomycin A1 (BafA1), a specific proton pump inhibitor that blocks Hepatitis C virus (HCV) infection through raising endosomal pH. These results demonstrate that immobilized HCV pseudoparticles (HCVpp) and HCVcc enter susceptible cells through a receptor- and low pH-dependent pathway, providing a convenient model to study the function of E1E2
The exact roles of the four cellular factors required for HCV entry and the spatiotemporal regulation of E1E2-mediated fusion have not been fully elucidated
Experimental findings of this study reveal that, contrary to the view that CD81 is a secondary HCV receptor, this protein plays a central role in entry of this virus

Summary

Introduction

We show that HCV pretreatment with an acidic buffer or with the soluble CD81 fragment increased infectivity, whereas sequential incubation with the receptor and low pH in the absence of a target membrane irreversibly diminished the ability of the virus to enter permissive cells. HCV Infection Is Enhanced by Pretreatment with the CD81 Extracellular Loop—The lack of HCVpp and HCVcc inactivation following the acidic pretreatment implies that E1E2 becomes competent for low pH-mediated fusion only after interactions with one or several cellular receptors, perhaps similar to the priming step of ASLV envelope glycoprotein by its receptor [41].

Results

Conclusion