A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses
Figure 9
HCV abrogated Riplet-mediated RIG-I activation.
(A and B) The inhibition of IFN-β promoter activation by NS3-4A was assessed by reporter gene assays. IPS-1-C508A mutant protein harbors an amino acid substitution at Cys-508 with Ala. 100 ng of IPS-1, IPS-1-C508A, RIG-I, Riplet, NS3-4A, and/or NS3-4A* expression vectors were transfected into HEK293 cells in 24-well plates with p125luc reporter plasmid. The total amount of transfected DNA (800 ng/well) was kept constant by adding empty vector (pEF-BOS). 24 hours after the transfection, the reporter activities were measured. Data are presented as mean ± SD (n = 3). *p<0.05. (C and D) IPS-1 KO mouse hepatocyte was transfected with IPS-1 C508A, RIG-I, Riplet, and/or NS3-4A expression vectors together with p125luc and Renilla luciferase plasmids. Transfected cells were stimulated with 50 ng of HCV dsRNA for 24 hours by transfection (D). Data are presented as mean SD (n = 3). *p<0.05. (E and F) Intracellular localizations of endogenous TBK1 and RIG-I were determined by confocal microscopy. HepG2, HuH7, and HuH7.5 cells were stimulated with 100 ng of HCV dsRNA for six hours by transfection (E). Stimulated cells (E) and O cells with HCV replicons (F) were stained with anti-RIG-I, TBK1, and/or NS3 antibodies. (G and H) HuH7 (G) and HuH7.5 (H) cells were infected with HCV JFH1 strain. Seven days after the infection, the cells were stained with anti-RIG-I, IPS-1, and NS3 antibodies. (I) HuH7 cells were infected with SeV at MOI = 1 for 24 hours. Cell lysates were prepared from mock or SeV infected HuH7 or HuH7 cells with HCV replicons (O cell). Immunoprecipitation using high salt buffer was performed with anti-RIG-I (Alme-1) antibody. The samples were subjected to SDS-PAGE. Endogenous K63-linked polyubiquitin chain was detected using ubiquitin K63-linkage specific antibody. (J) HuH7 cells were infected with SeV at MOI = 1 for 24 hours. Cell lysates were prepared from mock or SeV infected HuH7 or HuH7 cells with HCV replicons (O cell). Immunoprecipitation was performed with anti-RIG-I (Alme-1) antibody. The samples were subjected to SDS-PAGE. (K) HuH7 cells were transfected with siRNA for mock or Riplet. 48 hours after the transfection, cells were infected with HCV JFH1 for 2 days. RT-qPCR was performed to determine HCV genome RNA, GAPDH, and Riplet expression.