Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein

Abstract

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to “native” E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.

Flunarizine prevents hepatitis C virus membrane fusion in a genotype‐dependent manner by targeting the potential fusion peptide within E1

“…The E1 domain from residue 264-290 has previously been implicated in harboring an HCV fusion peptide based on phylogenetic comparison with related flaviviruses, sequence conservation, and hydrophobicity. 25 The observation that drugs that specifically inhibit membrane fusion select for viral resistance in this region indirectly supports the notion that this peptide is critical for fusion. The crystal structures of E2 did not reveal evidence for a major role of E2 as a fusion protein, so E1 is now assumed to be the primary fusion protein of HCV.…”

Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion

“…Finally, structural analyses at low pHs indicate that E2 does not undergo the structural rearrangement needed to play a role in membrane fusion (13). On the other hand, a conserved highly hydrophobic region in E1 (residues 264 to 290) has been proposed previously as a potential HCV fusion loop (60). The importance of this region in viral infectivity has been confirmed by several mutagenesis studies (15,61,62).…”

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

“…While the hybrid proteins were indeed transported to the cell surface, their incorporation into MV particles was below the limit of detection. In contrast, analogous hybrid glycoproteins expressed from vesicular stomatitis virus (10,63) and influenza virus (23) vectors are readily detected in viral particles. We do not know why this difference exists, but in our experimental system, based on an infectious MV cDNA that is vaccine equivalent, foreign glycoprotein incorporation may be more stringent.…”