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
. 2014 Jan 7;7(307):ra3.
doi: 10.1126/scisignal.2004577.

The ubiquitin-specific protease USP15 promotes RIG-I-mediated antiviral signaling by deubiquitylating TRIM25

Eva-Katharina Pauli  1 , Ying Kai Chan, Meredith E Davis, Sebastian Gableske, May K Wang, Katharina F Feister, Michaela U Gack
Affiliations

The ubiquitin-specific protease USP15 promotes RIG-I-mediated antiviral signaling by deubiquitylating TRIM25

Eva-Katharina Pauli et al. Sci Signal. .

Abstract

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys(63))-linked ubiquitin chains to the RNA sensor retinoic acid-inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys(48)-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys(48)-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I-dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I-mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. USP15 interacts with TRIM25
(A) HEK 293T cells were transfected with plasmids encoding GST or GST-TRIM25-RING-BB together with plasmid encoding USP15-V5. Forty-eight hours later, cells were lysed and whole-cell lysates (WCLs) were subjected to GST pull-down (GST-PD) and Western blotting (IB) analysis with anti-V5 antibody (α-V5) and anti-GST antibody (α-GST). Molecular mass markers (kD) are indicated to the left of the blots. (B) HEK 293T cells were left uninfected or were infected with SeV (50 HA U/ml). WCLs were prepared and subjected to immunoprecipitation (IP) with anti-USP15 antibody (α-USP15), and samples were analyzed by Western blotting with anti-TRIM25 antibody (α-TRIM25) and α-USP15 antibody. (C) Domain structures of TRIM25 and USP15, as well as a schematic representation of the GST-fused or Myc-tagged truncation mutants of TRIM25 and USP15, respectively. Numbers indicate amino acid residues. (D) HEK 293T cells were transfected with plasmids encoding the Myc-tagged DUSP, UBL, or His-Box domains of USP15 together with plasmids encoding GST or GST-TRIM25-RING-BB. Forty-eight hours later, WCLs were prepared and subjected to GST pull-down and Western blotting analysis with anti-Myc antibody (α-Myc) and α-GST antibody. WCLs were also analyzed by Western blotting with α-Myc. The upper part of this panel was exposed for a longer time than was used for the lower part. (E) HEK 293T cells were transfected with plasmids encoding GST or the indicated GST fusion constructs together with plasmid encoding Myc-tagged USP15 His-Box. Forty-eight hours later, WCLs were prepared and subjected to GST pull-down analysis, which was followed by Western blotting analysis with α-Myc and α-GST antibodies. Data in all panels are representative of three independent experiments.
Fig. 2
Fig. 2. USP15 deubiquitylates TRIM25
(A) HEK 293T cells were transfected with empty plasmid or with plasmid encoding TRIM25-FLAG together with plasmids encoding V5-USP15 wild type (WT) or the V5-USP15 C783A mutant. Forty-eight hours later, WCLs were prepared and subjected to immunoprecipitation with α-FLAG antibody and Western blotting analysis with anti-ubiquitin (α-Ub) and α-FLAG antibodies. WCLs were further used for Western blotting with α-V5 and anti-actin (α-actin) antibodies. (B) HEK 293T cells were transfected with control plasmid or with plasmid encoding USP15-Myc. Thirty hours later, cells were mock-treated or were infected with SeV (50 HA U/ml) for 16 hours. Ubiquitylation of endogenous TRIM25 was determined by immunoprecipitation of samples with α-TRIM25 antibody followed by Western blotting analysis with α-Ub antibody. (C and D) HEK 293T cells were transfected with empty plasmid or plasmid encoding TRIM25-FLAG together with (C and D) nontargeting control siRNA (si.C), (C) USP15-specific siRNA (si.USP15), or (D) USP11-specific siRNA (si.USP11). Forty-eight hours later, WCLs were subjected to immunoprecipitation with α-FLAG and Western blotting analysis with α-Ub and α-FLAG. The extent of knockdown of USP15 or USP11 was determined by Western blotting analysis of WCLs with the appropriate antibodies. (E) An in vitro deubiquitylation assay was performed with TRIM25-FLAG and the indicated amounts of purified USP15 protein. Nonubiquitylated and monoubiquitylated forms of TRIM25 were detected by Western blotting analysis with α-FLAG. The input amounts of USP15 protein were determined by Western blotting analysis with α-USP15. Data in all panels are representative of three independent experiments.
Fig. 3
Fig. 3. USP15 stabilizes TRIM25 by counteracting its ubiquitylation by LUBAC
(A) HEK 293T cells were transfected with plasmid encoding TRIM25-FLAG together with empty plasmid or plasmid encoding USP15-V5, as well as a plasmid encoding a HA-tagged ubiquitin (Ub) mutant in which all of the lysines except Lys48 (K48) are mutated (HA-K48only-Ub). Thirty-six hours later, cells were treated with 50 μM MG132 for 6 hours. WCLs were subjected to immunoprecipitation with α-FLAG antibody, and samples were then analyzed by Western blotting with α-HA and α-FLAG antibodies. (B) HEK 293T cells were transfected with plasmid encoding TRIM25-FLAG with or without plasmids encoding HOIP-Myc and HOIL-1L-V5, as well as with empty plasmid or plasmids encoding V5-tagged USP15 WT or the USP15 C783A mutant. Mono- and polyubiquitylation of TRIM25 were determined by immunoprecipitation with α-FLAG antibody and Western blotting analysis with α-Ub antibody. The presence of USP15, HOIL-1L, HOIP, and actin was determined in WCLs by Western blotting analysis with α-V5, α-Myc, and α-actin antibodies. (C) Ectopically expressed USP15 enhances the stability of TRIM25. HeLa cells were transfected with empty plasmid or plasmid encoding Myc-tagged USP15. Forty hours later, cells were treated with 100 μM cycloheximide (CHX) for the indicated times. Total protein amounts in WCLs were determined by bicinchoninic acid (BCA) assay, and equivalent amounts of WCLs were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting analysis with α-TRIM25, α-Myc, and α-actin antibodies. Data in all panels are representative of at least three independent experiments. Data from the densitometric analysis of three experiments represented by the blots in (B) and (C) are presented in figs. S4 and S5, respectively.
Fig. 4
Fig. 4. USP15 enhances TRIM25- and RIG-I–mediated signaling
(A and B) HEK 293T cells were transfected with the pGK–β-gal plasmid [which constitutively expresses β-galactosidase (β-gal)] and either (A) IFN-β or (B) NF-κB reporter plasmids, together with plasmids encoding GST–RIG-I(2CARD) and TRIM25-V5 and increasing amounts of plasmid encoding USP15-Myc. Forty-eight hours later, luciferase and β-gal values were determined as described previously (16). Data are means ± SD of duplicate samples and are representative of four independent experiments. (C and D) IFN-β luciferase activity in HEK 293T cells that were transfected with the indicated constructs was determined as described in (A). Data are means ± SD of duplicate samples and are representative of (C) three or (D) two independent experiments. (E) Measurement of IFN-β luciferase activity in HEK 293T cells transfected with plasmid encoding GST–RIG-I 2CARD alone or together with plasmid en-coding TRIM25-FLAG and that stably expressed either nontargeting control shRNA (sh.C) or a USP15-specific shRNA (sh.USP15). Luciferase and β-gal values were determined 48 hours after transfection. Data are means ± SD of duplicate samples and are representative of three independent experiments. (F) Twenty-eight hours after transfection with the indicated siRNAs together with the IFN-β luciferase and pGK–β-gal plasmids, HEK 293T cells were mock-treated or were infected with SeV (5 HA U/ml). Luciferase and β-gal values were determined 20 hours later. Data are means ± SD from three independent experiments. **P < 0.01. (G) HEK 293T cells, stably expressing sh.USP15 or sh.C, were infected with SeV (50 HA U/ml) for 16 hours. The extent of ubiquitylation of endogenous RIG-I was then determined by immunoprecipitation with α–RIG-I antibody and Western blotting analysis with α-Ub antibody. Data are representative of three independent experiments.
Fig. 5
Fig. 5. USP15 is required for the RIG-I– and TRIM25-mediated production of IFN and an effective antiviral response
(A) HEK 293T cells were transfected with empty plasmid or with plasmid encoding USP15-Myc. Thirty-six hours later, the cells were mock-treated or were infected with SeV (25 HA U/ml). Sixteen hours later, the abundances of IFN-β, ISG15, USP15, and actin mRNAs were determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. (B and C) Primary NHLFs were transfected with si.C or si.USP15. Forty-eight hours later, cells were left uninfected or were infected with SeV [5 HA U/ml (left) or 25 HA U/ml (right)]. The amounts of IFN-β protein secreted by the cells were determined by enzyme-linked immunosorbent assay (ELISA) analysis of culture medium ~17 hours after infection. *P < 0.05. N.D. indicates nondetectable. (C) Knockdown of USP15 was confirmed by Western blotting analysis. (D) Forty-eight hours after transfection with the indicated constructs, HEK 293T cells were infected with VSV-eGFP (at an MOI of 0.01). Twenty-seven hours later, eGFP was detected by fluorescence microscopy, and viral titers were determined by plaque assay. PFU, plaque-forming units. Data are representative of four independent experiments. Western blotting analysis was performed to detect the indicated USP15 proteins. (E) HEK 293T cells, stably expressing sh.C or sh.USP15, were infected with VSV-eGFP (MOI, 10−5). Twenty-four hours later, eGFP-positive cells were visualized by fluorescence microscopy and quantified by flow cytometry. Knockdown of endogenous USP15 was confirmed by Western blotting analysis. **P < 0.01. (F) HEK 293T cells transfected with the indicated siRNAs were infected with NDV-GFP (MOI 0.01). Twenty-seven hours later, GFP-positive cells were determined by flow cytometry. Knockdown of USP15 was confirmed by Western blotting analysis. **P < 0.01. Data in bar graphs in (B), (E), and (F) are means ± SD from three independent experiments. Other data are representative of at least three independent experiments.
Fig. 6
Fig. 6. Model for the activation of TRIM25 and RIG-I signaling by USP15
(Left) Upon viral infection, binding of RIG-I to viral RNA induces a conformational change in RIG-I that enables TRIM25 to bind to CARD1 in RIG-I and stimulate the Lys63-linked ubiquitylation of the CARD2, which leads to its subsequent binding to MAVS. (Middle) Later during viral infection, expression of the genes encoding LUBAC is induced. LUBAC then mediates the Lys48-linked ubiquitylation of TRIM25, which leads to its proteasomal degradation. (Right) The action of LUBAC is antagonized by USP15, which removes Lys48-linked polyubiquitin from TRIM25, leading to the stabilization of TRIM25 and a sustained antiviral IFN response.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci. 2008;1143:1–20. - PubMed
    1. Nakhaei P, Genin P, Civas A, Hiscott J. RIG-I-like receptors: Sensing and responding to RNA virus infection. Semin Immunol. 2009;21:215–222. - PubMed
    1. Hornung V, Ellegast J, Kim S, Brzoóka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G. 5′-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314:994–997. - PubMed
    1. Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, Reis e Sousa C. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science. 2006;314:997–1001. - PubMed
    1. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5:730–737. - PubMed

Publication types

MeSH terms