Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry

Abstract

BackgroundEnvelope (E) glycoprotein E2 of the hepatitis C virus (HCV) mediates binding of the virus to target cell receptors. Nevertheless, the precise role of E1 in viral entry remains elusive.MethodsTo understand the involvement of the fusion peptide-like domain positioned at residues 264 to 290 within envelope glycoprotein E1 in HCV infection, mutants with Ala and Asn substitutions for residues conserved between HCV and E proteins of flaviviruses or the fusion proteins of paramyxoviruses were constructed by site-directed mutagenesis and their effects on membrane fusion and viral infectivity were examined.ResultsNone of these mutations affected the synthesis or cell surface expression of envelope proteins, nor did they alter the formation of a non-covalent E1-E2 heterodimer or E2 binding to the large extracellular loop of CD81. The Cys residues located at positions 272 and 281 were unlikely involved in intra- or intermolecular disulfide bond formation. With the exception of the G267A mutant, which showed increased cell fusion, other mutants displayed reduced or marginally inhibited cell fusion capacities compared to the wild-type (WT) E1E2. The G267A mutant was also an exception in human immunodeficiency virus type 1 (HIV-1)/HCV E1E2 pseudotyping analyses, in that it showed higher one-cycle infectivity; all other mutants exhibited greatly or partially reduced viral entry versus the WT pseudotype. All but the G278A and D279N mutants showed a WT-like profile of E1E2 incorporation into HIV-1 particles. Since C272A, C281A, G282A, and G288A pseudotypes bound to Huh7 cells as effectively as did the WT pseudotype, the reduced infectivity of these pseudotypes was due to their ability to inhibit cell fusion.ConclusionOur results indicate that specific residues, but not the structure, of this fusion peptide-like domain are required for mediating cell fusion and viral entry.

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

“…It has been proposed that a conserved hydrophobic sequence, CSALYVGDLC (residues 272 to 281), in E1, which was not included in the structural analysis of nE1, may represent a truncated class II fusion peptide (27). The role of this sequence in virus entry and membrane fusion was supported by data from several studies (27)(28)(29)(30)(31)(32). The potential role of this putative fusion peptide in E1 in other aspects of the HCV life cycle has not been explored.…”

“…It was reported previously that a region (amino acid residues 272 to 285) ( Fig. 5A) in HCV E1 is a putative FP, which plays an important role in triggering the fusion of the viral envelope and endosomal membrane during the HCV entry process (27)(28)(29)(30)(31)(32). We first examined the role of FP using lentivirus-based HCV pseudotype particles (HCVpp) that harbor the HCV envelope proteins E1 and E2 and mimic the HCV entry process (36,37).…”

“…The recently solved crystal structure of the core domain of E2 failed to identify any fusion-triggering domains/motifs in E2 (21,22,26). It has been proposed that E1 may contain a putative FP (27)(28)(29)(30)(31)(32). Recently the 3.5-Å-resolution crystal structure of nE1 (residues 192 to 271) was solved (23).…”

The deletion of residues 268-292 of E1 impairs the ability of HCV envelope proteins to induce pore formation

“…Mutation analysis of hydrophobic residues in the putative fusogenic region of E1 has revealed that only the F285A mutant abolished viral entry whereas other mutations did not affect or only partially inhibited viral entry (Drummer et al, 2007). Finally, it has been reported that specific residues between positions 264 and 290 within E1, but not the structure, are responsible for fusion (Li et al, 2009). …”

Computational Prediction of the Heterodimeric and Higher-Order Structure of gpE1/gpE2 Envelope Glycoproteins Encoded by Hepatitis C Virus

“…The model also shows that C272 from nE1 is very close to C281 in the putative fusion peptide of E1, suggesting a potential for interactions. However, Li et al (65) noted that it is unlikely that these two residues are disulfide linked, since E1/E2 from HCV pseudoparticles (HCVpp) with the mutations C272A and C281A did not migrate differently on reducing or nonreducing SDS-PAGE gels compared to wild-type E1/E2. It is unclear based on experimental data in the literature which, if any, disulfide linkages are formed between E1 and E2 within the heterodimer and in the higher-order structure.…”