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. 2013;9(8):e1003533.
doi: 10.1371/journal.ppat.1003533. Epub 2013 Aug 8.

A distinct role of Riplet-mediated K63-Linked polyubiquitination of the RIG-I repressor domain in human antiviral innate immune responses

Hiroyuki Oshiumi  1 , Moeko Miyashita, Misako Matsumoto, Tsukasa Seya
Affiliations

A distinct role of Riplet-mediated K63-Linked polyubiquitination of the RIG-I repressor domain in human antiviral innate immune responses

Hiroyuki Oshiumi et al. PLoS Pathog. 2013.

Abstract

The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Riplet promotes TRIM25-mediated full-length RIG-I activation.
(A, B) Wild-type and Riplet KO MEFs were infected with SeV at MOI = 0.2. The mRNA expressions of IFN-β (A) and –α2 (B) at the indicated times after viral infection were determined by RT-qPCR. Results are presented as mean ± SD (n = 3). (C, D) The activation of IFN-β promoter was examined by reporter gene assay using a p125luc IFN-β reporter. RIG-I CARDs (C) or full-length RIG-I (D) expression vectors were transfected into HEK293 cells together with a Riplet and/or TRIM25 expression vector as indicated. 24 hours after the transfection, the reporter activities were determined. (E, F) The activation of IFN-β promoter was examined by reporter gene assay using a p125luc IFN-β reporter. Full-length RIG-I expression vectors were transfected into HEK293 cells together with a Riplet and/or TRIM25 expression vector as indicated. 24 hours after the transfection, the cells were stimulated with 50 ng of HCV 3′ UTR dsRNA by transfection or infected with VSV at MOI = 1 for 24 hours, and then reporter activation was determined. Data are presented as mean ± SD (n = 3). *p<0.05. (G) HEK293FT cells were stimulated with 0.8 µg of HCV double-stranded RNA (HCV RNA) using lipofectamine 2000 in 6-well plate. Cell lysates were prepared at the indicated times, followed by immunoprecipitation with an anti-RIG-I mAb (Alme-1).
Figure 2
Figure 2. Riplet and TRIM25 ubiquitin ligases associate with RIG-I.
(A, B) HeLa cells were transfected with Riplet-HA expression vector (A) or FLAG-Riplet and TRIM25-HA (B). 24 hours after transfection, the cells were infected with VSV at MOI = 1 for six hours. The cells were fixed and stained with anti-RIG-I (Alme-1), HA, and/or FLAG antibodies as indicated. Histograms display the measured fluorescence intensity along the white line in the merged panels. (C–F) Colocalization coefficients of Riplet localization to RIG-I (C) or TRIM25 (E) in mock or VSV infected HeLa cells. Pearson's correlation coefficient of Riplet and RIG-I (D) and TRIM25 (F) (mean ± SD, n>10). (G, H) HeLa cells were transfected with Riplet-HA (G) or TRIM25-HA (H) expression vector. The cells were stimulated with 100 ng of short polyI:C for six hours. The cells were fixed and stained with anti-G3BP and HA antibodies. Colocalization coefficient indicates values (mean ± SD, n>10) of Riplet (G) or TRIM25 (H) localization to G3BP staining region. Histograms display the measured fluorescence intensity along the white line in the merged panels. (I, J) TRIM25 (I) or Riplet (J) expression vector was transfected into HEK293FT cells together with FLAG-tagged-RIG-I CARDs or -RIG-I RD expression vectors. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG antibody. (K) Riplet, RIG-I, and/or RIG-I-ΔRD, which lacks RD, expression vector was transfected into HEK293 cell with p125luc reporter. Reporter activation was determined at 24 hours after transfection. Data are presented as mean ± SD (n = 3).
Figure 3
Figure 3. Riplet function is dispensable for RIG-I RNA binding activity.
(A) Expression vectors encoding Riplet, FLAG-tagged RIG-I CARDs, and/or FLAG-tagged RIG-I RD were transfected into HEK293FT cells together with an HA-tagged ubiquitin expression vector. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG (RIG-I) antibody. (B) HEK293FT cells were transfected with expression vectors encoding FLAG-tagged RIG-I, Riplet, and HA-tagged ubiquitin. Cell lysate was prepared at 24 hours after transfection, and then incubated with biotin-conjugated (Biotin-dsRNA) or non-conjugated (dsRNA) double-stranded RNA. Biotin-dsRNA was pull-downed with streptavidin beads. Samples were subjected to SDS-PAGE, and proteins were detected by western blotting. (C) HEK293FT cells were transfected with FLAG-tagged wild-type RIG-I, RIG-I 5KR, or RIG-I K788R expression vector. 24 hours after the transfection, the cell lysate was prepared. The pull down assay with biotin-dsRNA was performed as described above.
Figure 4
Figure 4. Lys residues within RIG-I RD were essential for Riplet-mediated K63-linked polyubiquitination.
(A) Schematic diagram of RIG-I mutant proteins. (B–D) IFN-β promoter activation was determined using a p125luc IFN-β reporter gene. HEK293 cells were transfected with 0.1 µg of expression vectors encoding full-length RIG-I, RIG-I K172R, RIG-I 5KR, and RIG-I K788R in 24-well plate. The transfected cells were stimulated with 0.1 µg of HCV 3′ UTR dsRNA using lipofectamine 2000 reagent for eight hours (C) or infected with VSV at MOI = 1 for 24 hours (D), after which reporter activation was determined. Data are presented as mean ± SD (n = 3). *p<0.05. (E) The expression vectors encoding HA-tagged Riplet and FLAG-tagged RIG-I, RIG-I 5KR, and RIG-I K788R were transfected into HEK293FT cells together with Myc-tagged K63-only ubiquitin expression vector, in which all Lys residues except Lys-63 within ubiquitin were replaced with Arg. At 24 hours after transfection, cell lysates were prepared, followed by immunoprecipitation with an anti-FLAG (RIG-I) antibody. (F) The expression vectors encoding FLAG-tagged wild-type and mutant RIG-I proteins were transfected into HEK293FT cells together with HA-tagged ubiquitin expression vector. 24 hours after the transfection, the cells were stimulated with HCV 3′ UTR dsRNA by transfection for eight hours. Then cell lysate was prepared and immunoprecipitation was performed with anti-FLAG antibody.
Figure 5
Figure 5. Riplet affects RIG-I RD autorepression of CARDs signaling.
(A, B) IFN-β promoter activation was determined using a p125luc IFN-β reporter gene. Expression vectors encoding RIG-I CARDs, full-length RIG-I, RIG-I K788R, RD, RD-K788, and/or Riplet were transfected into HEK293 cells as indicated. Reporter activation was determined after transfection. Data are presented as mean ± SD (n = 3). *p<0.05. (C) IFN-β promoter activation was determined using a p125luc IFN-β reporter gene. RIG-I and Riplet expression vectors were transfected into HEK293 cells as indicated. 24 hours after transfection, cells were infected with SeV at MOI = 1 for 24 hours, and the reporter activities were determined. (D–F) HEK293FT cells were transfected with expression vectors encoding FLAG-tagged RIG-I, HA-tagged TRIM25, HA-tagged Riplet, FLAG-tagged RIG-I K788R as indicated. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG antibody (RIG-I). Relative band intensity of immunoprecipitated TRIM25 in panel D was determined (E). (G) Mouse splenocyte was isolated from wild-type and Riplet KO mice spleen. The cells were infected with SeV, and then cell lysate was prepared. Immunoprecipitation was carried out with anti-RIG-I rabbit mAb (D14G6), and subjected to SDS-PAGE. Endogenous K63-linked polyubiquitin chain was detected using K63-linked polyubiquitin chain specific antibody.
Figure 6
Figure 6. The association of TBK1 and IKK-ε protein kinases with RIG-I RD is enhanced by Riplet.
(A–F) The interaction of RIG-I with TBK1, IKK-ε, and NEMO was examined by immunoprecipitation assay. FLAG-tagged RIG-I or RIG-I RD expression vector was transfected into HEK293FT cells together with HA-tagged IKK-ε (A, B, and E), Myc-tagged TBK1 (C, F), HA-tagged NEMO ubiquitin binding region (D), and/or Riplet (D–F) expression vectors as indicated. 24 hours after the transfection, cell lysate was prepared, and immunoprecipitation was performed with anti-FLAG antibody. Asterisk indicates non-specific bands. (G) Relative band intensity of immunoprecipitated NEMO, IKK-ε, and TBK1 in D–F was determined. (H–L) Intracellular localization of endogenous RIG-I (H–J), TBK1 (H–J), phosphorylated-TBK1 (p-TBK1) (K), and mitochondria (I–K) were observed using anti-RIG-I (Alme-1), TBK1, p-TBK1 mAbs, and mitotracker. HeLa cells were stimulated with HCV dsRNA for six hours using lipofectamine 2000. Colocalization coefficient of TBK1 localization to RIG-I (H), RIG-I and TBK1 localization to mitochondria (I), and TBK1 and p-TBK1 localization to mitochondria (L) were determined (mean ± sd, n>10). Person's correlation coefficient of RIG-I and TBK1 was determined (H). (M) Splenocytes from wild type and Riplet KO mouse were infected with VSV at MOI = 10 for eight hours. Immunoprecipitation was performed using an anti-RIG-I rabbit monoclonal antibody (D14G6), and the immunoprecipitates were analyzed by SDS-PAGE. Endogenous RIG-I, TBK1, TRIM25, and β-actin were detected using anti-RIG-I, p-TBK1, TRIM25, and β-actin antibodies.
Figure 7
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.
Figure 8
Figure 8. NS3-4A inhibits Riplet-mediated RIG-I polyubiquitination.
(A, B) Riplet, NS3-4A, and/or HA-tagged ubiquitin (HA-Ub) expression vectors were transfected into HEK293FT cells along with either full-length RIG-I (A) or RIG-I RD (B). Cell lysate was prepared 24 hours after transfection, and subjected to SDS-PAGE. The proteins were detected by western blotting. (C). HEK293FT cells were transfected with Myc-tagged K63-only ubiquitin, FLAG-tagged RIG-I RD, and/or NS3-4A expression vectors. 24 hours after the transfection, cells were infected with SeV for six hours, and then cell lysate was prepared. Immunoprecipitation was carried out with anti-FLAG antibody, and the samples were subjected to SDS-PAGE. (D) HA-tagged TRIM25, Riplet and/or FLAG-tagged RIG-I expression vectors were transfected into HEK293FT cells with or without NS3-4A expression vector. Cell lysate was prepared 24 hours after the transfection, and immunoprecipitation assay was performed with anti-FLAG antibody. The precipitates were subjected to SDS-PAGE.
Figure 9
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.
Figure 10
Figure 10. Model for Riplet-mediated RIG-I activation.
In resting cell, RIG-I RD represses its CARDs-mediated signaling. When RIG-I CTD associates with viral RNA, Riplet mediates K63-linked polyubiquitination of RIG-I RD, leading to the association with TRIM25 and TBK1. K63-linked polyubiquitin chain mediated by TRIM25 induces RIG-I oligomerization and association with IPS-1 adaptor. TBK1 associated with RIG-I is activated on mitochondria.
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This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, and the Ministry of Health Labour, and Welfare of Japan, Kato Memorial Bioscience Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.