FIG 2.

RIG-I deletion in Huh7 cells does not restore HCV NS4A Y16F replication. Huh-7.5 cells (A) or Huh7 cells (B) were infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Immunoblot analysis was performed on lysates extracted at the indicated hours postinfection (hpi) or after mock infection (M). Graphs directly to the right of each blot (here and in panel F) show quantification of NS5A protein relative to tubulin at 72 hpi (mean ± the SEM; n = three biological replicates). (C) IFNB1 expression relative to HPRT1 from Huh7 cells infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 1) at 72 hpi as analyzed by RT-qPCR, with data displayed as means ± the SEM (n = three biological replicates). (D) Immunoblot of extracts of Huh7 and Huh7-RIG-I KO cells that were mock or SV infected (20 h). (E) IFN-β promoter reporter luciferase expression of Huh7 and Huh7-RIG-I KO cells expressing either vector or full-length RIG-I that were either mock or SV infected (20 h). Values show means ± the SD (n = three technical replicates) in RLU. (F) Huh7-RIG-I KO cells were infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Immunoblot analysis was performed on lysates extracted at the indicated times or mock infected (M). The graph directly to the right of this blot shows the quantification of NS5A protein relative to tubulin at 72 hpi (mean ± the SEM; n = 3 biological replicates). (G to I) Focus-forming assay of supernatants harvested from Huh-7.5 (G), Huh7 (H), and Huh7-RIG-I KO (clones 1 and 2) (I) cells at 72 hpi (MOI of 0.3). The data are presented as the percent HCV titer from the Y16F strain relative to the WT (set at 100%) and show means ± the SEM (n = two or three biological replicates). Data were analyzed by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.005; NS, not significant).