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. 2018 Apr 17;46(10):5139–5158. doi: 10.1093/nar/gky273

Figure 4.

Figure 4.

miR-122 binding does not shield the 5′ terminus of HCV RNA against RLR recognition. Huh-7 cells were electroporated with siRIG-I or siControl (siCon) at day –3 and at day 0 cells were electroporated again with the indicated siRNA and either (A) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA, or (B) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. (C) Western blot showing knockdown efficiency with antibodies against RIG-I and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. Huh-7 cells were electroporated with siMDA5 or siControl (siCon) at day –3, at day 0 cells were electroporated again with the indicated siRNA, and either (D) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or (E) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. (F) Quantitative PCR analysis indicating knockdown efficiency using MDA5-specific and GAPDH-specific TaqMan probes. MDA5 mRNA levels were calculated relative to the siCon. (G) Huh-7 cells were electroporated with siMDA5 on day –3, treated with 50 IU/ml IFN-α on day –1 and harvested for western blot at day 0 using antibodies against MDA5 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. (H) Northern blot analysis of FL HCV RNA accumulation during MDA5 knockdown at day 3. (I) Densitometry quantification of northern blot analysis in (H) normalized to siCon. Huh-7 cells were electroporated with siLGP2 or siControl (siCon) at day –2, at day 0 cells were electroporated again with the indicated siRNA, and either (J) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or (K) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. (L) Western blot showing knockdown efficiency with antibodies against LGP2 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. All data are representative of at least three independent experiments and statistical significance was determined by paired parametric t test.

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