Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection

Abstract

Hepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction. IMPORTANCEHepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

An Entropic Safety Catch Controls Hepatitis C Virus Entry and Antibody Resistance

“…However, at lower, physiological, temperatures the intrinsic fluorescence ratio of I438V A524T and ΔHVR-1 E2 were higher than WT (Figure S10A & 4E), consistent with mutant and ΔHVR-1 E2 being biophysically distinct from WT. Notably, HVR-1 is directly upstream of a highly conserved tryptophan residue in AS412 that has been previously demonstrated to be important for CD81 binding (W420, (Cowton et al, 2016; Owsianka et al, 2006)).…”

Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data

“…Certain residues seem to be key target residues as they are recognized by several bNAbs from different sources these include; W420 in Epitope 1 and F442 in Epitope 2 that are targeted by 12 and 11 bNAbs, respectively (Figure 2 ). We have shown that W420 is a critical residue modulating interactions with the cellular receptors CD81 and SR-BI ( 31 ). Other residues only form part of the epitope for 1 bNAb, for instance, T416, A439, and D533.…”

A polymorphic residue that attenuates the antiviral potential of interferon lambda 4 in hominid lineages

“…Luciferase-expressing MLV pseudoparticles containing the E1 and E2 glycoproteins from JFH1 HCV strain were generated as described [ 68 ] along with their corresponding JFH1 E1-E2 deficient controls (particles generated only with MLV core) and used to challenge IFNλ-stimulated Huh7 cells. Huh7 cells grown in 96-well plates overnight (seeded at 4 x 10 3 cells per well) were stimulated with IFNλs for 24 hrs and transduced with HCVpp.…”