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. 2019 Jun 28;93(14):e00693-19. doi: 10.1128/JVI.00693-19

FIG 2.

FIG 2

Sam68 promotes HCV replication without affecting viral translation. (A) FL-neo or FCA1 cells were transfected with increasing amounts of plasmid encoding Flag-tagged Sam68 for 48 h. Huh7.5 or HLCZ01 cells were infected with HCV at an MOI of 0.1 for 12 h and then transfected with an increasing amount of pFlag-tagged Sam68 for 60 h. All cell lysates were detected by immunoblotting with the indicated antibodies. β-Actin was used as an internal control. Triangles indicate increasing amounts of plasmid. (B) FL-neo or FCA1 cells were transfected with either the control shRNA or the Sam68 shRNA plasmid for 48 h. Huh7.5 or HLCZ01 cells were preinfected with HCV at an MOI of 0.1 for 12 h and then transfected with either the control shRNA plasmid or the Sam68 shRNA plasmid for 60 h. All cell lysates were detected by the indicated antibodies. (C) Huh7.5 cells expressing either scrambled shRNA (sh-Ctrl) or Sam68-targeting shRNA (sh-Sam68) were infected with HCV for 72 h followed by immunostaining using an anti-HCV NS5A antibody (red). DAPI was used to counterstain nuclei (blue). (D) The intracellular HCV RNA was measured by real-time PCR assay (normalized to GAPDH); meanwhile, the cells were under the same treatment as Fig. 2A. *, P < 0.05; **, P < 0.01. (E) FL-neo or FCA1 cells were transfected with a gradient of the Sam68 shRNA plasmid (1 μg, 2 μg) for 48 h. Huh7.5 or HLCZ01 cells were preinfected with HCV at an MOI of 0.1 for 12 h and then transfected with a gradient of the Sam68 shRNA plasmid (1 μg, 2 μg) for 60 h. The intracellular HCV RNA was analyzed by real-time PCR and normalized to GAPDH. (F, G) Huh7.5 (F) or HLCZ01 (G) cells were transfected with the indicated plasmids for 24 h together with the pRL-HL for an additional 24 h. Cell extracts were harvested to conduct a dual-luciferase reporter assay. (H, I) Huh7.5 cells were transfected with pFlag-vector or pFlag-Sam68 or control shRNA or Sam68 shRNA plasmid and then electroporated with the in vitro transcribed RNA (SGR-Luc-JFH1-GND [H] or SGR-Luc-JFH1 [I]). The luciferase assay was performed at the indicated time point postelectroporation.