Fig 2.

CPIs interfere with HCV RNA association with the NS5A complex. (A) Input HCV RNA levels from JFH-FLAG-infected cells exposed to various treatments for 8 h. CsA was used at 1,000 ng/ml, IFN was used at 10 U/ml and 40 U/ml, and BMS-790052 was used at 10 pM. The HCV RNA was normalized to the amount of GAPDH mRNA present in each lysate, and the untreated sample value was set to 1. (B) CsA treatment significantly reduces NS5A-associated HCV RNA. Each lysate was subjected to FLAG-IP followed by RNA recovery from IP samples and analysis by qRT-PCR. The untreated sample value was set to 1. (C) Dose-dependent inhibition of RNA binding of NS5A complex by CsA. (Left) HCV RNA detected in FLAG-IP complexes isolated from JFH-FLAG-infected cells treated with CsA at the indicated concentrations for 8 h. Percentages of precipitated RNA versus input RNA were calculated, and the untreated sample value was set to 1. (Right) Western blot analysis of NS5A levels in treated cell lysates. (D) Analysis of RNA binding by NS5A of the JFH-FLAG-DEYN virus. The RNA and NS5A protein were analyzed as described for panel C.