Figure 7. NS3-4A of HCV targets the Riplet protein.

(A–D) Endogenous RIG-I and Riplet protein levels were observed by western blotting. HeLa cells were stimulated with polyI:C transfection (A), HCV dsRNA transfection (B) or infected with VSV (C). HCV replicon positive (HCV) and negative (-) cell lysates were prepared from a HuH7-derived cell line O cell that contains HCV 1b full-length replicons and O curred cell (Oc cell) in which HCV replicons were removed by IFN treatment (D). (E) The response to HCV RNA in wild-type and Riplet KO MEFs was examined by RT-qPCR. Wild type (WT) and Riplet knockout (KO) MEF cells were transfected with 100 ng of HCV ssRNA and dsRNA. Six hours after stimulation, mRNA expressions of IFN-α2, IP10, and IFN-λ2/3 were measured by RT-qPCR. Data are presented as mean ± SD (n = 3). *p<0.05. (F–H) FLAG-tagged Riplet and RIG-I (F), HA-tagged Riplet (G), or HA-tagged TRIM25 (H) expression vectors were transfected into HEK293FT cells together with NS3-4A or NS3-4A* expression vectors. NS3-4A* mutant protein harbors an amino acids substitution at its catalytic site Ser-139 with Ala. 24 hours after transfection, cell lysate was prepared and subjected to SDS-PAGE. (I) Band intensity ratio of IPS-1, Riplet, TRIM25, IKK-ε, and Riplet-3A with/without NS3-4A expression (mean ± sd, n = 3). (J) NS3-4A cleavage sites within an HCV polypeptide are compared with a candidate site in the Riplet RING-finger domain. Homologous amino acids are shown in bold, and identical amino acids are underlined. In Riplet-3A mutant protein, three acidic amino acids, Glu-16, Asp-17, Asp-18, were substituted with Ala. (K) An expression vector encoding wild-type Riplet or Ripled-3A mutant protein was transfected into HEK293 cells together with NS3-4A or NS3-4A* expression vectors. Cell lysate was prepared 24 hours after transfection, and subjected to SDS-PAGE. (L) HA-tagged Riplet-3A and NS3-4A expression vectors were transfected into HepG2 cell. 24 hours after transfection, the cells were fixed and stained with anti-HA monoclonal antibody (mouse) and anti-NS3-4A polyclonal antibody (goat). (M) N-terminal FLAG-tagged Riplet was expressed in HEK293FT cells, and immunoprecipitation was carried out with anti-FLAG antibody. Immunoprecipitates were incubated with recombination NS3-4A purified from E.coli at 37°C for one hour, and samples were subjected to SDS-PAGE analysis. The proteins were detected by western blotting. (N) Purified GST fused Riplet (1–210 aa) was incubated with or without recombinant NS3-4A (rNS3-4A) at 37°C for 30 min. The proteins were subjected to SDS-PAGE and detected by western blotting.