Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Oct 28;105(43):16743-8.
doi: 10.1073/pnas.0804947105. Epub 2008 Oct 23.

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

Michaela U Gack  1 , Axel Kirchhofer, Young C Shin, Kyung-Soo Inn, Chengyu Liang, Sheng Cui, Sua Myong, Taekjip Ha, Karl-Peter Hopfner, Jae U Jung
Affiliations

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

Michaela U Gack et al. Proc Natl Acad Sci U S A. .

Abstract

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA in a 5'-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDs of RIG-I undergo the K(63)-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-I with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-I first and second CARD in TRIM25-mediated RIG-I ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T(55)I mutation in RIG-I first CARD abolishes TRIM25 interaction, whereas the K(172)R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-I function is further evidenced by a RIG-I splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36-80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-I multimerization and RIG-I-MAVS signaling complex formation, this SV effectively suppresses the RIG-I-mediated IFN-beta production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-I activation but also identifies the RIG-I SV as an off-switch regulator of its own signaling pathway.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The intact tandem CARD of RIG-I is essential for TRIM25-mediated RIG-I activation. (A and B) TRIM25 interaction with RIG-I first CARD. HEK293T cells were transfected with GST, GST-RIG-I 2CARD, GST-RIG-I first CARD, or GST-RIG-I second CARD together with TRIM25-V5 (A) or TRIM25-SPRY-V5 (B). Whole-cell lysates (WCLs) were subjected to GST-pulldown (GST-PD), followed by immunoblotting (IB) with α-V5 or α-GST. Arrows indicate the ubiquitinated bands. (C) Both CARDs are necessary for RIG-I ubiquitination and interaction with MAVS. At 48 h after transfection with GST, GST-RIG-I 2CARD, GST-RIG-I first CARD, or GST-RIG-I second CARD together with MAVS-CARD-PRD-Flag, HEK293T WCLs were used for GST-PD, followed by IB with α-GST, α-Ub, or α-Flag. (D) Both CARDs are necessary for TRIM25-mediated RIG-I signaling. GST-RIG-I fusion constructs with or without TRIM25 together with IFN-β luciferase and constitutive β-gal-expressing pGK-β-gal were expressed in HEK293T cells. Luciferase and β-galactosidase values were determined as described (20). Data represent the mean ± SD (n = 3).
Fig. 2.
Fig. 2.
The T55 residue of RIG-I is critical for TRIM25 binding. (A) T55I mutation abolishes RIG-I-TRIM25-interaction. At 48 h after transfection with GST, GST-RIG-I 2CARD WT, or GST-RIG-I 2CARD T55I together with TRIM25-V5 WCLs were used for GST-PD, followed by IB with α-V5 or α-GST. Arrows indicate the ubiquitinated bands. (B and C) T55I mutation abolishes RIG-I CARD ubiquitination and downstream signaling. GST-RIG-I 2CARD WT or GST-RIG-I 2CARD T55I with or without TRIM25-V5 was expressed in HEK293T. WCLs were used for GST-PD, followed by IB with α-GST or α-Ub. Arrows indicate the ubiquitinated bands. (C) GST-RIG-I 2CARD WT or T55I with or without TRIM25 together with IFN-β luciferase and pGK-β-gal were expressed in HEK293T cells as described in Fig. 1D. Data represent the mean ± SD (n = 3). (D) T55I mutation strongly decreases RIG-I binding to MAVS. HEK293T were transfected with MAVS-CARD-PRD-Flag together with GST or GST-RIG-I 2CARD fusion constructs. WCLs were subjected to GST-PD, followed by IB with α-Flag, α-Ub, or α-GST. MAVS-CARD-PRD expression was determined by IB with α-Flag. (E and F) Ubiquitination, TRIM25 binding, and signaling activity of RIG-I 2CARD T55 mutants. GST or GST-RIG-I 2CARD was expressed in HEK293T cells. WCLs were subjected to GST-PD, followed by IB with α-Ub, α-GST, or α-TRIM25. Arrows indicate the ubiquitinated bands. Luciferase assay was performed as described in Fig. 1D. Data represent the mean ± SD (n = 3).
Fig. 3.
Fig. 3.
Identification and biochemical characterization of RIG-I SV. (A) Schematic protein representations of RIG-I full-length and SV. (B) Transcript (Upper) and protein (Lower) of RIG-I SV. HEK293T cells were mock-treated or stimulated with IFN-β (1,000 units/ml) for the indicated number of hours. Total RNA was subjected to RT-PCR to amplify the RIG-I CARDs (exon 1–3). Transcript levels of RIG-I and RIG-I SV were further determined by using specific primers. The actin transcript was used as a control. RIG-I full-length and SV levels were determined with α-RIG-I. (C) Lack of TRIM25 binding and CARD ubiquitination of RIG-I SV. After transfection with GST, GST-RIG-I 2CARD WT, or GST-RIG-I 2CARD SV with or without TRIM25-V5, HEK293T WCLs were used for GST-PD, followed by IB with α-GST, α-Ub, or α-V5. Arrows indicate the ubiquitinated bands. (D) RIG-I SV does not interact with MAVS. At 48 h after transfection with MAVS-CARD-PRD-Flag and GST-RIG-I 2CARD, HEK293T WCLs were used for GST-PD, followed by IB with α-Flag, α-Ub, or α-GST. (E) Lack of signaling activity of RIG-I SV. HEK293T cells were transfected with GST, GST-RIG-I 2CARD WT, or GST-RIG-I 2CARD SV with or without TRIM25-V5 together with IFN-β luciferase and pGK-β-gal. Data represent the mean ± SD (n = 3). (F) Abolished antiviral function of RIG-I SV. RIG-I, RIG-I SV, or RIG-I T55I mutant was stably expressed in RIG-I−/− MEFs, and these cells were infected with VSV-eGFP at MOI 0.5. At 40 h after infection, virus titer and replication were determined by plaque assay and GFP expression, respectively. Pfu, plaque-forming unit.
Fig. 4.
Fig. 4.
RIG-I SV inhibits RIG-I-mediated antiviral signaling. (A) RIG-I SV inhibits the SeV-induced IFN-β promoter activation. HEK293T were transfected with vector or increasing amounts of RIG-I T55I or RIG-I SV together with IFN-β luciferase and pGK-β-gal. At 24 h after transfection, cells were mock-treated or infected with SeV, and luciferase activity was determined as described in Fig. 1D. Data represent the mean ± SD (n = 3). RIG-I SV inhibits virus-induced IRF3 phosphorylation (B) and dimerization (C). (B) At 24 h after infection with Myc-RIG-I (10 μg), Flag-RIG-I SV (2 μg) or Flag-RIG-I T55I (2 μg), cells were mock-treated or infected with SeV (50 HA units/ml) for 16 h. WCLs were subjected to IB with α-Phospho-IRF3 (Ser396), α-IRF3, α-Myc, α-Flag, or α-actin. (C) At 24 h after transfection with Flag-IRF3, Flag-RIG-I WT, RIG-I SV, or RIG-I T55I, cells were mock-treated or infected with SeV (50 HA units/ml) for 18 h. WCLs were used for native PAGE and subjected to IB with α-IRF3. (D) RIG-I SV inhibits the virus-induced nuclear translocation of IRF3. At 24 h after transfection with IRF3-eGFP, HEK293T stably expressing vector, Flag-RIG-I, Flag-RIG-I SV, or Flag-RIG-I T55I were mock-treated or infected with SeV and stained with α-RIG-I (red) and Hoechst 33256 (nucleus). (E) RIG-I SV expression increases VSV-eGFP replication. HEK293T stably expressing vector, RIG-I, RIG-I SV, or RIG-I T55I were infected with VSV-eGFP at MOI 0.5. At 24 h after infection, virus titer and replication were determined by plaque assay and GFP expression, respectively. (F) RIG-I SV suppresses virus-induced IFN-β production. HEK293T stably expressing vector, RIG-I, RIG-I SV, or RIG-I T55I were infected with SeV (50 HA units/ml) for 20 h. IFN-β production was measured by ELISA. Data represent the mean ± SD (n = 3).
Fig. 5.
Fig. 5.
RIG-I splice variant inhibits virus-induced RIG-I multimerization and RIG-I-MAVS complex formation. (A) The important role of the C-terminal RD of RIG-I splice variant for its inhibitory activity. After transfection with vector, RIG-I SV, or its mutants (K270A or C810,813A) together with IFN-β luciferase and pGK-β-gal, HEK293T cells were mock-infected or infected with SeV (50 HA units/ml) for 15 h. Data represent the mean ± SD (n = 3). (B) RIG-I splice variant interacts with RIG-I WT. After transfection with Flag-RIG-I WT, Flag-RIG-I SV, or Flag-RIG-I T55I together with Myc-RIG-I WT, HEK293T were mock-infected or infected with SeV (50 HA units per ml) for 15 h and WCLs were used for IP with α-Myc, followed by IB with α-Flag. (C) RIG-I splice variant interferes with virus-induced RIG-I multimerization. Upon expression of Flag-RIG-I SV or RIG-I T55I, Myc-RIG-I WT multimerization was determined by IB with α-Myc in native PAGE. To detect RIG-I monomer, the lower part of the membrane was exposed for a longer period of time (second from top). (D) RIG-I splice variant inhibits RIG-I-MAVS-interaction. HEK293T were cotransfected with MAVS-CARD-PRD-Flag and Myc-RIG-I WT together with vector, V5-RIG-I SV, or V5-RIG-I T55I and subsequently either mock-treated or infected with SeV (50 HA units per ml) for 15 h. WCLs were subjected to IP with α-Myc, followed by IB with α-Flag.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hiscott J, Lin R, Nakhaei P, Paz S. MasterCARD: A priceless link to innate immunity. Trends Mol Med. 2006;12:53–56. - PubMed
    1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783–801. - PubMed
    1. Meylan E, Tschopp J, Karin M. Intracellular pattern recognition receptors in the host response. Nature. 2006;442:39–44. - PubMed
    1. Creagh EM, O'Neill LA. TLRs, NLRs and RLRs: A trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol. 2006;27:352–357. - PubMed
    1. Hornung V, et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314:994–997. - PubMed

Publication types