The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types
Fig 2
HCVcc mutants with modified p7 amino-termini prevent infectivity.
(A) Schematic representation of JFH1 and Jc1 HCVcc mutants in E2-p7 junction. The first helix of p7 is shown in the light grey box. The insertions of residues are shown in dark grey and the substitutions are underlined. (B-D) Western blot analysis of Huh7.5 cells electroporated with RNAs from HCVcc viruses encoding wild-type or E2p7 cleavage mutants. (B) At 72h post-electroporation, cells were lysed and digested with endoH glycosidase before western blotting using 3/11 antibodies against E2. (C) The relative ratios of E2p7 precursors vs. free E2 species are indicated. (D) The HAHALp7 protein and the E2HAHALp7 (white arrows) and E2HAHALp7NS2 (black arrows) precursors were revealed using an HA tag antibody in lysates of cells electroporated with the indicated HCVcc constructs. (E) The extracellular (black) and intracellular (grey) infectivity levels for all mutants are represented. (F) The extracellular (black) and intracellular (grey) infectivity levels, normalized after determining the proportion of HCV-positive virus producer cells (see Fig 3A), of JFH1 HAHALp7 or p7-T2 mutant viruses expressed alone or with wild-type p7 (+ p7) are represented relative to infectivity of parental JFH1 virus. (G) The extracellular (black) and intracellular (grey) infectivity levels, normalized by determining the proportion of HCV-positive virus producer cells, of Jc1 HAHALp7 mutant virus expressed alone or with wild-type p7 or HAHALp7, as indicated, are represented relative to infectivity of parental Jc1 virus. Data are displayed as means ± SEM. The numbers of experiments performed are indicated below the graphs.