FIG 5.

The impact of miR-122 binding at site 1 compared to binding at site 2 with full-length replicons in Hep3B cells. (A) Because Hep3B cells lack expression of detectable miR-122, we used them to test the influence of miR-122 binding to each site separately or together in both S1:p3 and S2:p3 mutant HCV RNAs through supplementation with the appropriate miRNAs, either miR-122 or miR-122/p3 (i through iv). (B) A time course of virus replication in Hep3B cells electroporated with full-length S1:p3 RNA (or the indicated control viral RNAs: FL WT + miR-122, S1+S2:p3 + miR-122/p3 or miControl, and FL GNN) and supplemented as indicated with miR-122, miR-122/p3, both, or miControl, to achieve miR-122 binding site combinations depicted in panel Aiii. Replication was measured by Renilla luciferase expression at the indicated time points. (C) The effect of binding at miR-122 site S1 or S2 on replication of FL S1:p3 HCV RNA is shown relative to luciferase expression from unbound full-length viral RNA, S1+S2:p3 [S1+S2 Unbound], dotted line, at 3 days postelectroporation. Significance for relevant comparisons was determined by unpaired parametric t test. (D) A time course of virus replication in Hep3B cells electroporated with full-length S2:p3 RNA (or the indicated control viral RNAs: FL WT + miR-122, S1+S2:p3 + miR-122/p3 or miControl, and FL GNN) and supplemented with miRNAs as indicated to achieve miR-122 binding site combinations shown in panel Aiv. Replication was measured by Renilla luciferase expression at the indicated time points. (E) The effect of binding at miR-122 site S1 or S2 on replication of FL S2:p3 HCV RNA is shown relative to luciferase expression from unbound full-length viral RNA, S1+S2:p3 [S1+S2 Unbound], dotted line, at 3 days postelectroporation. Significance for relevant comparisons was determined by unpaired parametric t test.