Analysis of a conserved RGE/RGD motif in HCV E2 in mediating entry

Abstract

Background: Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors. It is clear that HCV uses a multi-receptor complex to gain entry into susceptible cells, however key elements of this complex remain elusive. In this study, the role of a highly conserved RGE/RGD motif of HCV E2 glycoprotein in viral entry was examined. The effect of each substitution mutation in this motif was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E2 proteins, CD81 binding profiles, and conformation of mutants were examined.

Results: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 90% of wt HCVpp], CD81 binding, E1E2 expression, and incorporation into viral particles and proper conformation) or very low infectivity (< or = 20% of wt HCVpp). Only amino acid substitutions of the 3rd position (D or E) resulted in wt characteristics as long as the negative charge was maintained or a neutral alanine was introduced. A change in charge to a positive lysine, disrupted HCVpp infectivity at this position.

Conclusion: Although most amino acid substitutions within this conserved motif displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, and incorporation into HCVpp. Our results suggest that although RGE/D is a well-defined integrin binding motif, in this case the role of these three hyperconserved amino acids does not appear to be integrin binding. As the extent of conservation of this region extends well beyond these three amino acids, we speculate that this region may play an important role in the structure of HCV E2 or in mediating the interaction with other factor(s) during viral entry.

Figure 1

Conserved RGE or RGD motif of hepatitis C virus E2. HCV strains from the Los Alamos HCV sequence database were aligned. The conserved RGE or RGD motif is boxed in red. Amino acids are numbered relative to the AUG start codon of the H77 strain (boxed in black) used in this study.

Figure 2

Amino acid substitutions within the conserved RGE motif of E2 dramatically affect hepatitis C virus pseudoparticle entry. 293 T cells were cotransfected with HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 48 h PI and used to infect Huh7 cells. Infectivity was measured 72 h PI using a luciferase reporter assay. Numbers shown above the bars are infectivity of each mutant expressed as a percentage of the infectivity observed for the wild-type (wt) H77 E1E2. Values shown are the mean and standard error for a minimum of three assays.

Figure 3

Expression and incorporation of hepatitis C virus E1E2 glycoproteins in producer cell lysate and HCVpp. (A) 293 T producer cells were lysed on analyzed by Western blot analysis using anti (α)-E2. Detection of actin was performed as a loading control. (B) Incorporation of HCV glycoproteins into HCVpp was determined by pelleting the virus through a 20% sucrose cushion followed by Western blot analysis. Detection of p24 capsid protein was performed as a loading control.

Figure 4

Blocking RGE/RGD sites on Huh7 cells prior to hepatitis C virus pseudoparticle challenge. (A) Huh7 cells were incubated at 4°C for 1 h with ECM proteins then washed and infected with HCVpp. (B) Inhibition of HCVpp infectivity by peptides GRGDTP, GRGESP, RGD-E2 or RGE-E2 was assessed by incubating Huh7 cells with increasing amounts of peptide at 4°C for 1 h, washing and infecting with HCVpp. Infecitivity for both was determined 72 h PI using a luciferase reporter assay.

Figure 5

Binding of hepatitis C virus E2 glycoproteins containing RGE substitutions to soluble CD81. 293 T cells transfected with HCV E1E2 wt of mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with soluble CD81-GST fusion protein. Binding to CD81 was determined by Western blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt.

Figure 6

Conformation of HCV E2 glycoprotein substitutions within conserved RGE/RGD motif. 293 T cells transfected with HCV E1E2 wt or RGE/RGD mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. HCV E2 R614A was used as a negative control.