(A) Huh7.25.CD81 cells were transfected for 24 hrs with 150 ng of the pGL2-IFNβ-FLUC; 40 ng of the pRL-TK-RLUC reporter plasmids alone or in presence of 150 ng of a plasmid expressing HA-TRIM25, either as such (TRIM25wt) or containing the P₃₅₈L substitution (a SNP rs75467764 with no reported pathology). Cells were infected or not with SeV (40 HAU/ml) for 24 hrs. IFN expression was expressed as fold induction of luciferase activity. Error bars represent the mean ± S.D. for triplicates. (B) Huh7, Huh7.5 and Huh7.25.CD81 cells were either untreated or treated with 500 U/ml IFNα for 24 hrs. TRIM25 was detected by immunoblot after preparation of nuclear and cytosolic fractions from 25 µg of cell extracts. Detection of Actin, RIG-I and phosphorylated Histone 3 (HH3-P) served as controls. (C–D) Huh7.25.CD81 cells were transfected with the reporter plasmids as in A, a few hours before being treated with 500 U/ml IFNα for 24 hrs (C) or left untreated (C or D). They were then infected with Sendai virus (40 HAU/ml) or with JFH1 at an m.o.i of 0.2 (C) or increasing from 0.2 to 10 (D). At the times indicated, IFN expression was expressed as fold induction of luciferase activity. Error bars represent the mean ± S.D. for triplicates. Induction of TRIM25 after IFN treatment was shown by immunoblot (C). (E) The Huh7.25.CD81 cells were transfected for 48 hrs with 5 µg of His-Myc-Ubiquitin expression plasmid in absence or presence of a plasmid expressing HA-TRIM25 and infected with SeV (40 HAU/ml) or HCV (m.o.i = 6). At the times indicated, 10% of the lysate was precipitated with TCA and the remaining lysate subjected to nickel pulldown under denaturing conditions. Total and ubiquitin (Ub)-modified proteins were separated by SDS-PAGE and revealed by immunoblot.

(A–B). The Huh7.25.CD81 cells were transfected with 25 nM of the different siRNA (Control, ISG15, PKR), separately or together. After 48 hrs, cells were infected with JFH1 (m.o.i = 0.2). At the times indicated, expression of HCV or IFNβ RNA was determined by RTqPCR and expressed as copies of JFH1 RNA (A) or as fold induction (IFNβ; B). The expression levels of IFNβ RNA at the start of infection was 6.96×10⁵ copies. (C) Two sets of Huh7.25.CD81 cells were first transfected with siRNA ISG15, siRNA PKR separately and together for 24 hrs, then transfected with the reporter plasmids IFNβ-firefly luciferase (pGL2-IFNβ), pRL-TK Renilla-luciferase for another 24 hrs and infected with JFH1 (m.o.i = 0.2) for the times indicated. In each case, IFN expression was expressed as fold-induction over control cells that were simply transfected with pGL2-IFNβ-FLUC/pRL-TK-RLUC. The graph represents the level of firefly luciferase activity normalized to the ratio R-luc RNA/GAPDH RNA. Such normalization is required because of the negative control of general translation through PKR after 12 hrs post-infection [8]. Error bars represent the mean ±S.D for triplicates. (D) Huh7.25.CD81 cells, in 100 cm² plates, were transfected with siRNA Control or siRNA ISG15 or transfected with a plasmid expressing HA-ISG15 for 48 hrs and infected with JFH1 (m.o.i = 6). At the indicated times post-infection, cell extracts (2.2 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on two different NuPAGE gels and blotted using Mab 71/10 or anti-phosphorylated PKR antibodies (PKR-P). The presence of PKR and PKR-P was revealed using the Odyssey procedure. The ratio PKR-P/PKR in the absence or in the presence of ISG15, either endogenous or endogenous and ectopic, is shown in Figure S5.

(A–B). A-The Huh7.25.CD81 cells were transfected with 50 nM Control siRNA and the different Smartpool siRNAs (50 nM siPKR; 10 nM siRIG-I; 5 nM siMAVS; 50 nM siTRAF3; 50 nM siIRF3) for 48 hrs and infected with JFH1 (m.o.i = 6). (B) Huh7.5 or Huh7 cells were transfected with siRNA Control or siPKR (50 nM) for 48 hrs and infected with JFH1 (m.o.i = 0.2 for Huh7.5 or 10 for Huh7). At the times indicated, expression of endogenous ISG15 was determined by RTqPCR and expressed as fold induction. Error bars represent the mean ±S.D for triplicates. The expression level of ISG15 RNA at the start of infection in the siControl cells was 9.97×10⁴ copies (Huh7.25.CD81), 1.31×10⁴ copies (Huh7.5) and 1.28×10⁴ (Huh7). (C–D) Huh7.25.CD81 cells, in 100 cm² plates, were infected with JFH1 (m.o.i = 0.2) alone (C) or in presence of PRI or C16 (D). At the times indicated, cell extracts (3.5 mg) were processed for immunoprecipitation of PKR or for incubation with mouse IgG as a control of specificity (asterisk). The detection of the proteins in the complexes and in the whole cell extracts (WCE) was revealed by immunoblot using the Odyssey procedure. (E) The Huh7.25.CD81 cells were incubated with PRI or C16 and infected with JFH1 (m.o.i = 0.2) for the times indicated. Expression of endogenous ISG15 was determined as in A–B. The ISG15 RNA levels were 3.81×10⁴ copies in the siControl cells. (F) Human primary hepatocytes (HHP) were infected with JFH1 (m.o.i = 6). One set of cells was incubated with 30 mM of the PRI inhibitor during 8 hours. At the times indicated, expression of HCV RNA, ISG15 and IFNβ was determined by RTqPCR. The expression levels of ISG15 and IFNβ RNA at the start of infection was 1.05×10⁵ copies and 1,11×10⁴ copies, respectively. Inhibition of induction of ISG15 by PRI at 8 hr post-infection was statistically significant (***; p = 0.0001).

Soon after infection, the HCV RNA is detected by the dsRNA binding domains (DRBD) of PKR ahead (2 hr) of its recognition by the RNA helicase RIG-I (6 hr). Recruitment of PKR by HCV triggers a signaling pathway that involves PKR as an adapter protein to recruit MAVS and TRAF3. This leads to a strong induction of the di-ubiquitine like protein ISG15 as well as other IRF3-dependent ISGs (Interferon-Stimulated Genes). ISG15 negatively controls the TRIM25-mediated ubiquitination (Ub) of RIG-I through an ISGylation process and thus interferes with the ability of RIG-I to recruit its downstream partners, including MAVS and TRAF3, and to induce IFNβ and ISGs. As the infection proceeds, HCV activates the eIF2α kinase function of PKR (12 hr). This leads to a transient (few hours) inhibition of general translation, including that of IFN [8] and ISGs [9] while the eIF2α-independent translation of the viral proteins proceeds unabated. At later times in the infection (18 hr), additional control of IFN induction occurs through cleavage of MAVS by the HCV NS3/4A protease, once the viral proteins have sufficiently accumulated in the cytosol [7], [27].

(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.

(A–C) The cDNAs reversed transcribed from the RNAs extracted from the Huh7.25.CD81 cells for the experiment described under Figure 3A were analysed by qPCR for the expression of ISG15 (A) and ISG56 (B). Expression levels of ISG15 and ISG56 RNA at the start of infection were respectively 1×10⁵ and 1.18×10⁵ copies. A novel set of Huh7.25.CD81 cells were transfected with siRNA Control, siRNA ISG15, siRNA PKR separately and together for 48 hrs. They were then transfected with the reporter plasmids ISG56-FLUC and pRL-TK-RLUC and infected with JFH1 (m.o.i = 0.2). At the times indicated, the effect of the different conditions of silencing on the reporter expression was analyzed after normalization performed as described under Figure 3C (C). Results are expressed as fold induction. Error bars represent the mean ±S.D for triplicates. (D) Huh7.25.CD81 cells were either transfected with 25 nM of siRNA Control or siPKR for 24 hrs and infected with SeV for the times indicated. Expression of endogenous ISG15 was determined by RTqPCR and expressed as fold induction. Error bars represent the mean ±S.D for triplicates. The expression levels of ISG15 RNA at the start of infection were respectively 4.91×10⁴ copies (siControl) and 5.44×10⁴ copies (siPKR). (E) Huh7.25.CD81 cells were either transfected with 25 nM of siRNA Control or siISG15 and with 1 µg of a plasmid expressing PKR where indicated. After 48 hrs, the cells were infected with HCV (m.o.i = 6) for the times indicated. Expression of HCV, ISG15 and IFNβ RNA was determined by RTqPCR. Cell lysates prepared from cells treated in the same conditions but not infected were used to control expression of PKR and ISG15 by immunoblot.

(A–B)- Huh7.25.CD81 cells were transfected with 25 nM of siRNA Control (A) or 25 nM of siRNA ISG15 (B) for 48 hrs and infected with JFH1 (m.o.i = 0.2). At the times indicated, cell extracts (4.5 mg) were incubated with anti-MAVS antibodies. In addition, cell extracts prepared at 0 hr post-infection were incubated with mouse IgG as a control of specificity (asterisk). The immunoprecipitated complexes were run on three different NuPAGE gels and blotted using Mab 71/10, anti-MAVS, anti-RIG-I or anti-TRAF3 antibodies. The expression level of each protein was controlled in the total cell extracts. (C)- Huh7.25.CD81 cells were incubated with JFH1 (m.o.i = 6) for 2 hrs at 37°C or at 4°C in the absence or presence of 30 µM of PRI. This drug was applied one hour before the end of the incubation time. After washing the cells twice with PBS, the cells were further incubated for 2, 4 or 6 hrs at 37°C or at 4°C in the absence or presence of PRI (added every hour). The cell extracts were processed for crosslinking of RNA to proteins before lysis, as described in Materials and Methods and different immunoprecipitations were performed with antibodies directed against PKR, RIG-I or eIF2α. After extensive washing, the presence of HCV RNA linked to the immunocomplexes was analysed by RTqPCR and the presence of the proteins was verified by Western blot. Measure of HCV RNA in the cell extracts allowed to estimate its percentage of binding to PKR as 1.09%, 0.47% and 0.25% at 2, 4 and 6 hrs post-infection respectively, and its percentage of binding to RIG-I as 0.34% at 6 hrs post-infection.