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. 2022 Feb 23;96(4):e01903-21. doi: 10.1128/jvi.01903-21

FIG 7.

FIG 7

miR-122 has a small but significant influence on the maintenance of HCV replication. (A) To test for the influence of miR-122 on ongoing HCV, replication cells were supplemented with miR-122 or an miR-122 antagonist after the establishment of an infection. MiR-122-dependent replication was established by electroporating Huh 7.5 miR-122 KO cells with wild-type J6/JFH-1 (p7Rluc2a) or ΔE2 J6/JFH-1 (p7Rluc2a) RNA and miR-122. Then, 3 days later the cells were transfected with miR-122, anti-miR-122, or miControl to assess the influence of miR-122 supplementation and antagonization, and 2 days later luciferase activity was measured as an indication of viral RNA amplification. (B) To assess the impact of miR-122 supplementation on HCV RNA replicating independently of miR-122, replication of U4C/G28A/C37U ΔE2 J6/JFH-1 (p7Rluc2a) HCV infection was established in Huh 7.5 miR-122 KO cells without miR-122 and on day 3 was supplemented by transfection with either miR-122 or control microRNA. Luciferase activity was measured on day 2 posttransfection as an indication of viral propagation. (C) To assess the impact of miR-122 supplementation on cells stably supporting miR-122-independent HCV RNA, we used Huh 7.5 miR-122 KO cells electroporated with J6/JFH-1 Neo (p7Rluc2a) HCV and selected with G418. The cells were then electroporated with either control microRNA or miR-122, and luciferase activity was measured at different time points posttransfection. (D) To assess the impact of miR-122 antagonization on miR-122-dependent HCV replication, we electroporated Huh 7.5 wild-type (WT) cells with J6/JFH-1 Neo (p7Rluc2a) HCV RNA and selected with G418. Stable cells were then transfected with either control LNA or anti-miR-122 LNA, and luciferase activity was measured at different time points posttransfection. All data shown are the average of three or more independent experiments. Error bars indicate the standard deviation of the mean, and asterisks indicate significant differences versus the control microRNA/LNA. The significance was determined by using Student’s t test (ns, not significant; *, P < 0.033; **, P < 0.002; ***, P < 0.001).