Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: putative CD81 binding region 1 is not involved in CD81 binding

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 including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation 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 E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.

Results: Based on these characteristics, mutants either displayed wt characteristics (high infectivity [> or = 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (< or = 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.

Conclusion: Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474-492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522-551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612-619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry.

Figure 1

Conserved residues within the putative CD81 binding domains of E2. HCV strains from the Los Alamos HCV sequence database were aligned. Three regions previously implicated in CD81 binding were analyzed. Amino acids are numbered relative to the AUG start codon of the H77 strain shown and used in this study. The hyperconserved (black rectangles) targeted (asterisk) residues for alanine substitution are indicated.

Figure 2

Alanine substitutions within putative CD81 binding regions dramatically affect HCVpp entry. 293T cells were cotransfected with the HIV-luc packaging vector along with HCV E1E2 mutant expression plasmids. HCVpp was harvested at 24 h post-transfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.

Figure 3

Expression and incorporation of HCV E1E2 glycoproteins in producer cell lysate and HCVpp. (A) 293T HCVpp producer cells were lysed and analyzed by Western Blot analysis using anti (α)-E2 and (α)-actin antibodies. Image is a composite. (B) Incorporation of HCV glycoproteins into HCVpp was determined by pelleting the virus through a 20% sucrose cushion followed by Western Blot analysis. HCV glycoproteins were identified with (α)-E2 and (α)-E1 antibodies. Detection of the HIV p24 capsid protein with an anti-HIV p24 antibody was performed as a loading control. Image is a composite.

Figure 4

Binding of mutant HCV E1E2 glycoproteins to soluble CD81. (A) 293T cells transfected with HCV E1E2 wt or 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. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific 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. Image is a composite.

Figure 5

Determining association of E1E2 mutants. 293T cells were transfected with HCV E1E2 wt, mutant, or E1 alone glycoprotein expression plasmids. Cells were lysed and cleared cell lysate was incubated with anti (α)-E2 antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western Blot for E1 to determine if the E2 and E1 glycoproteins had formed dimers. Image is a composite.