Figure 7. I861T substitution in NS2 is the major selective resistance mutation identified.

(A) Selection of resistance-conferring mutations was performed. Huh7.5 cells were infected with media from Huh7.5 cells transfected with RNA from wild type (WT) or selected I861T viral clone. The effect of aptamer NS2-2 on the production of infectious WT or selected I861T mutated virus particles was tested as described in Figure 3D. Results are the average of three independent experiments. *P<0.05, **P<0.01 verse control cells. (B) I861T mutation had no effect on viral protein expression. Huh7.5 cells were infected with media from Huh7.5 cells transfected with RNA from wild type (WT) or selected I861T viral clone, followed with NS2-specific aptamer treatment for 72 hours. Protein was isolated and NS2 or NS5A was detected by western blot analysis. (C) I861T mutation escaped the disruption of NS2-2 aptamer on the interaction of NS2 with NS5A. Huh7.5 cells were treated as described in part B. Protein was isolated from the HCV-infected Huh7.5 cells with aptamer or library treatment and immunoprecipitated with antibodies against NS5A or mouse IgG conjugated with agarose beads respectively. The protein binding to the beads were boiled and subjected to SDS-PAGE. The protein was transferred onto PVDF membrane and then reacted with mouse monoclonal anti-NS2 or NS5A antibody and secondary antibodies.