Figure 2. HCV controls RIG-I ubiquitination through ISG15.

(A) Huh7.25.CD81 cells were transfected for 24 hrs with 25 nM of siRNA (Control or ISG15) and for another 24 hr with 5 µg of a His-Myc-Ubiquitin plasmid in absence or presence of 5 µg of a plasmid expressing HA-TRIM25. The cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, cell extracts were processed for analysis of RIG-I ubiquitination and the expression of the different proteins in the total cell extracts. (B) Huh7.25.CD81 cells were first transfected with siRNA Control (25 nM), si RNA ISG15 (25 nM), siRNA Ube1L (50 nM) or left untreated. After 24 hrs, the untreated cells were transfected with a plasmid expressing HA-ISG15 (500 ng) alone or in presence of plasmids expressing E1, E2 and E3 (1 µg each) while a set of cells transfected with siRNA Ube1L received plasmids expressing HA-ISG15, E2 and E3. After 24 hrs, the cells were infected with JFH1 (m.o.i = 6) for the times indicated. Stimulation of endogenous IFNβ RNA expression was determined by RTqPCR and expressed as fold induction. The degree of statistical significance is indicated by stars after calculation of the p-values (from left to right: 0.0005, 0.0076, 0.0003, 0.047 and 0.0023). (C–D) Huh7.25.CD81 cells, transfected with 25 nM of siRNA (Control or ISG15) for 48 hrs, were infected with JFH1 (m.o.i = 6) for the times indicated. Expression of IFNβ or HCV RNA, determined by RTqPCR, was expressed as fold induction (C; IFNβ) or as copies (D; HCV). Error bars represent the mean ±S.D for triplicates. Expression levels of IFNβ RNA at the start of infection were 2.1×10⁴ (siControl) and 4×10⁴ copies (siISG15). Supernatants collected at different times post-infection were used to infect fresh cells. After 24 hours, the RNAs were extracted from the cells and expression of HCV RNA was determined by RTqPCR. (E) Huh7.25.CD81 cells, transfected with 25 nM of siRNA (Control or ISG15) for 48 hrs, were infected with JFH1 for the times indicated. Cell extracts were analysed by immunoblot with Abs directed against ISG15, MAVS, the HCV NS3 and core proteins and Actin as loading control.