Recognition of Native Hepatitis C Virus E1E2 Heterodimers by a Human Monoclonal Antibody

Abstract

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.Hepatitis C virus (HCV) is the causal agent of hepatitis C, which is a major health problem worldwide (28). HCV is a positive-strand RNA virus (2) that belongs to the Flaviviridae family. Its genome encodes two membrane-associated envelope glycoproteins, E1 and E2, which are N glycosylated in their large N-terminal ectodomains and are anchored into membranes by their C-terminal transmembrane domains (31). These latter domains have been shown to be endoplasmic reticulum (ER) retention signals (5,7,17,20). When expressed in cell culture, the E1 and E2 glycoproteins assemble into noncovalently linked E1E2 heterodimers. These noncovalent E1E2 complexes have been proposed as functional subunits of the HCV particle. In addition, a significant amount of E1 and E2 is also present in high-molecular-weight, disulfide-linked aggregates, thought to result from a nonproductive folding pathway leading to misfolded protein complexes (for review see reference 31).Because of the lack of a suitable cell culture system for in vitro propagation of HCV and the unavailability of virions in sufficient quantities, truncated, secreted versions of E2 have been used as soluble surrogates for native virus particles. Indeed, the identification of CD81 as the putative cellular receptor for HCV is based on its binding to a truncated form of E2 (36). Intriguingly, intracellular forms of truncated E2, enriched for the presence of monomeric, nonaggregated E2, were found to bind CD81 with greater affinity than did the secreted forms (18, 26), suggesting that antigenic or structural differences exist between intracellular and secreted forms of the E2 glycoprotein. Several murine monoclonal antibodies (MAbs) have been shown to recognize conformation-dependent epitopes within E2. Studies using these antibodies (Abs) (including MAb H53) have provided additional insight into the conformational state of the envelope glycoproteins during intracellular processing and folding and have helped to define a native, prebudding form of t...

Hepatitis C Patient-Derived Glycoproteins Exhibit Marked Differences in Susceptibility to Serum Neutralizing Antibodies: Genetic Subtype Defines Antigenic but Not Neutralization Serotype

“…2). In agreement with previous studies (11), all of the clones generated detectable monomeric E2 protein with a molecular mass of approximately 60 kDa.…”

Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

“…Since HCV envelope glycoprotein subunits cooperate during their folding for the formation of E1E2 heterodimer (17,39,48), we wanted to determine whether our mutants affect the formation of the E1E2 heterodimer, which is the functional HCV glycoprotein unit in infected cells (6). We first analyzed the expression of HCV glycoproteins individually by SDS-PAGE under reducing and nonreducing conditions to determine whether the absence of a cysteine residue in E1 can lead to the formation of disulfide bond aggregates involving both E1 and E2.…”

Human Monoclonal Antibody to Hepatitis C Virus E1 Glycoprotein That Blocks Virus Attachment and Viral Infectivity

“…HeLa cells infected with wild-type vaccinia virus vWA (Fig. 2, lanes 1, 3, and 5) or wild-type vaccinia virus vWA plus vaccinia virus rHCV1-1488 (lanes 2, 4, and 6) were lysed 24 h postinfection and were immunoprecipitated with H-111, CBH-2 (a conformation-dependent HMAb to HCV E2 protein [5,10], used as a positive control), or R04, an isotype-matched negative control, as described previously (11). The precipitated proteins were separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis under reducing conditions, and immunoblots were analyzed with rat anti-E2 MAb 3/11 (generously provided by J. McKeating) (8) (Fig.…”