HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation
Figure 3
Basic residues R50, K51, R59 and R62 in core are essential for NS5A binding.
A) Pulldowns using GST-Core122-170-His (negative control), wild-type GST-Core-BD2-His (wt) or single alanine GST-Core-BD2-His mutants (R50A, K51A, R59A, and R62A) as bait to capture full-length NS5A-His. Bound proteins were analyzed by Western blotting using anti-NS5A and anti-GST antibodies. Input (5%) used for each GST pulldown was probed with anti-His antibodies. The membrane was visualized using respective IRDye secondary antibodies and bound material quantified on the Odyssey IR imaging system. Amount of captured NS5A-His was expressed as IR signal of bound NS5A normalized to the amount of precipitated GST-Core proteins in each lane from three independent experiments. B) Flag co-immunoprecipitations in SGR-JFH1 cells transfected with an expression vector for wild-type 3xFlag-Core (wt), single alanine mutants (R50A, K51A, R59A, and R62A) or empty pcDNA3 plasmid. After immunoprecipitation with anti-Flag antibodies, bound material was eluted with 3xFlag peptide and analyzed by Western blotting with anti-NS5A and anti-Core antibodies. Input of whole-cell lysate (2%) used for each co-immunoprecipitation was probed with anti-NS5A, anti-Core and anti-Actin antibodies. The membrane was visualized using respective IRDye secondary antibodies and bound material quantified on the Odyssey IR imaging system. Amount of co-immunoprecipitated NS5A was expressed as IR signal of bound NS5A normalized to the amount of immunoprecipitated 3xFlag-Core proteins and protein expression levels in each lane from three independent experiments. C) Pulldowns using GST-Core122-170-His (negative control), wild-type GST-Core-BD2-His (wt) or double, triple and quadruple alanine core mutants (listed in Figure 1B) as bait to capture full-length NS5A-His. Bound proteins were analyzed by Western blotting as described above (A). D) Flag co-immunoprecipitations in SGR-JFH1 cells transfected with wild-type 3xFlag-Core (wt), double, triple and quadruple alanine core mutants (listed in Figure 1B) or empty pcDNA3 plasmid. Co-immunoprecipitated proteins were analyzed by Western blotting as described above (B).