FIG 1.

Surf4 participates in HCV replication. (A) SGR2a cells were transfected with siS-1 (10 nM, 25 nM, and 50 nM), siDHCR (25 nM), or siNT (25 nM). At 3 days posttransfection, inhibition of HCV replication (top blot) and silencing of proteins (middle and bottom blots) were determined by luciferase and immunoblot assays, respectively. The luciferase activity is shown in relative light units (RLU). The positions of molecular mass positions are shown in kilodaltons (kD) to the right of the blots. (B) SGR2a cells were treated with siSurf4, including siS-1, siS-2, and siS-3, or with siDHCR or siNT. Three days later, HCV replication was determined by luciferase assay. (C) Huh7 cells were transfected with pSilencer-shSurf4 or negative-control shNC and selected using hygromycin B. Knockdown of Surf4 in stable transfectants (shSurf4) was determined by immunoblotting. (D and E) shSurf4 and shNC cells were transfected with plasmids expressing Surf4shr, Surf4, or empty vector as indicated. After 24 h, transfected cells were electroporated with SGR2a RNA (D) or JFH1 genomic RNA (E). Three days later, intracellular HCV RNA was determined by real-time RT-PCR (graphs), and Surf4 expression was determined by immunoblotting. (F) Effect of Surf4 silencing on SGR1b. SGR1b cells were transfected with siSurf4 (25 nM), siDHCR (25 nM), or siNT (25 nM). At 3 days posttransfection, inhibition of HCV replication (graph) and silencing of proteins (blots) were determined by luciferase and immunoblot assays, respectively. (G) SGR2a, SGR1b, and Huh7 (control) cells were subjected to immunoblot assays using anti-Surf4, anti-NS5A, and antiactin antibodies. The relative intensity of each Surf4 band after normalization to that of actin is indicated by the number above the lane. Data in the graphs are averages of triplicate values with error bars representing standard deviations (SDs). Values that are significantly different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01.