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. 2004 Jun;78(12):6370–6380. doi: 10.1128/JVI.78.12.6370-6380.2004

FIG. 3.

FIG. 3.

Identification of the region responsible for processing of the signal sequence of HCV core protein by SPP and signal peptidase. (A) The hydrophobicity profile of HCV core protein was predicted by the method of Kyte and Doolittle (20). Hope and McLauchlan separated the HCV core protein into three regions, domains 1 to 3 (12). Two hydrophobic regions are predicted in the regions from amino acid 128 to 151 and from amino acid 164 to 186 in the C terminus of the HCV core protein. Mutations and deletions in the region from amino acid 128 to 151 of Flag-Core-HA and Flag-Core-E1-HA constructs are indicated. Dots indicate unchanged amino acids. (B) Expression of Flag-Core-HA polyproteins with changes of Ala191 to Arg in 293T cells. Flag-Core-HA (lanes 2 and 11), Flag-Core L139A-HA (lane 3), Flag-Core V140A-HA (lane 4), Flag-Core L144A-HA (lane 5), Flag-Core LV139/140AA-HA (lane 6), Flag-Core LL139/144AA-HA (lane 7), Flag-Core VL140/144AA-HA (lane 8), Flag-Core LVL/3A-HA (lane 9), and Flag-Core Δ128-151-HA (lane 10) were analyzed by immunoblotting with anti-Flag (upper panel) or anti-HA (lower panel) antibody. Cells transfected with an empty plasmid were used as a negative control (lane 1). (C) Expression of Flag-Core-E1-HA mutants in 293T cells. Flag-Core-E1-HA (lanes 2 and 11), Flag-Core L139A-E1-HA (lane 3), Flag-Core V140A-E1-HA (lane 4), Flag-Core L144A-E1-HA (lane 5), Flag-Core LV139/140AA-E1-HA (lane 6), Flag-Core LL139/144AA-E1-HA (lane 7), Flag-Core VL140/144AA-E1-HA (lane 8), Flag-Core LVL/3A-E1-HA (lane 9), and Flag-Core Δ128-151-E1-HA (lane 10) were analyzed by immunoblotting with anti-Flag (upper panel) or anti-HA (lower panel) antibody. The asterisk indicates unprocessed Flag-Core Δ128-151. Cells transfected with an empty plasmid were used as a negative control (lane 1). (D) The deglycosylation procedure is described in Materials and Methods. After transfection, cell lysates were immunoprecipitated with anti-HA antibody and immunoprecipitates were digested with Endo H (upper panel) or PNGase F (lower panel). Following digestion, proteins were separated by SDS-polyacrylamide gel electrophoresis, and material from cells transfected with vector (lane 1), Flag-Core-E1-HA (lane 2), Flag-Core LVL/3A-E1-HA (lane 3), and Flag-Core Δ128-151-E1-HA (lane 4) was detected by blotting with anti-HA. Nontreated and Endo H- or PNGase F-treated samples are indicated by − and +, respectively. Asterisks indicate mouse IgG heavy chains.