TRIM29 Negatively Regulates the Type I IFN Production in Response to RNA Virus

Junji Xing, Ao Zhang, Laurie J. Minze, Xian Chang Li and Zhiqiang Zhang
J Immunol July 1, 2018, 201 (1) 183-192; DOI: https://doi.org/10.4049/jimmunol.1701569

Abstract

The innate immunity is critically important in protection against virus infections, and in the case of RNA viral infections, the signaling mechanisms that initiate robust protective innate immunity without triggering autoimmune inflammation remain incompletely defined. In this study, we found the E3 ligase TRIM29 was specifically expressed in poly I:C–stimulated human myeloid dendritic cells. The induced TRIM29 played a negative role in type I IFN production in response to poly I:C or dsRNA virus reovirus infection. Importantly, the challenge of wild-type mice with reovirus led to lethal infection. In contrast, deletion of TRIM29 protected the mice from this developing lethality. Additionally, TRIM29−/− mice have lower titers of reovirus in the heart, intestine, spleen, liver, and brain because of elevated production of type I IFN. Mechanistically, TRIM29 was shown to interact with MAVS and subsequently induce its K11-linked ubiquitination and degradation. Taken together, TRIM29 regulates negatively the host innate immune response to RNA virus, which could be employed by RNA viruses for viral pathogenesis.

Introduction

Innate immunity provides the first line of defense against invading pathogens. Activation of innate immunity requires the recognition of pathogen-associated molecular patterns by pattern-recognition receptors (PRRs) (1). Retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs), including RIG-I and melanoma differentiation–associated protein 5 (MDA5), function as cytoplasmic RNA sensors that recognize viral nucleic acid dsRNA released during virus replication (2). Once they have recognized the viral RNA, these RLRs could be activated and initiate a series of signaling events that lead to the production of type I IFNs (IFN-α and IFN-β) (3). IFN-α/β further activate downstream signaling pathways that lead to the transcriptional induction of a wide range of genes encoding antiviral products, and those antiviral products act together to elicit a cellular antiviral response through various mechanisms (4).

The RLR signaling pathway triggers innate immune responses against RNA viruses. Although RIG-I and MDA5 sense distinct types of viruses (5–7), they share the common adaptor protein MAVS (also known as Cardif, IPS-1, or VISA) to transmit signals (8–11), which leads to IFN-β production for initiation of the antiviral innate immune response (12). MAVS is critical in innate antiviral immunity as the sole adaptor for RLRs. MAVS regulation is essential for the prevention of excessive harmful immune responses. Given that MAVS coordinates signals from two independent central sensors, RIG-I and MDA5, various mechanisms are employed to modulate MAVS, including protein–protein interactions, posttranslational modifications, and changes of mitochondrial dynamics (13). Recently, several negative regulators for MAVS have been reported (14–16). However, the detailed molecular regulation mechanisms of MAVS activation are not clear.

Tripartite motif proteins (TRIM) consist of a large family of proteins containing a RING–B-box–coiled-coil motif followed by different C-terminal domains. TRIM family proteins control important cellular processes such as intracellular signaling in innate immunity and viral infection, transcriptional regulation, development, autophagy, and carcinogenesis (17). TRIM29 is structurally a member of the TRIM protein family and is a unique multifunctional protein for DNA damage responses (18), molecular cancer biomarker (19), antiviral innate immunity (20), cell adhesion/invasion in tumor (21–23), cell differentiation, and signal regulation for the canonical Wnt pathway (24). Previously, we have found that E3 ubiquitin ligase TRIM29 is highly and constitutively expressed in alveolar macrophages and functions to control the respiratory infections by influenza virus and Haemophilus influenzae through regulating negatively the innate immunity in the respiratory tract (20). Also, we have shown that TRIM29 is constitutively expressed in human airway epithelial cells and induced by dsDNA in human myeloid dendritic cells (mDCs) and promotes DNA virus infections by inhibiting innate immune response (25). Given that TRIM29 has E3 ligase activity and is a multifunctional protein in cellular processes, we continued to investigate its role in innate immunity and viral infections. In this study, we identify the E3 ligase TRIM29, specifically expressed in poly I:C–induced human mDCs, as a critical negative regulator of the innate immune responses to RNA virus through targeting MAVS for degradation. Thus, our report reveals a previously unrecognized regulatory function of TRIM29 in the context of RNA virus infection in human mDCs.

Materials and Methods

Mice

Trim29 knockout (KO) (Trim29−/−) mice were from the European Mouse Mutant Archive (EM: 07120). Primary bone marrow was collected from wild-type (WT) and Trim29−/− mice for inducing mature bone marrow–derived dendritic cells (BMDCs) and bone marrow–derived macrophages (BMDMs) (20). All animals were maintained in the specific pathogen-free facility at Houston Methodist Research Institute in Houston, Texas. Animal use and care were approved by the Houston Methodist Animal Care Committee, in accordance with institutional animal care and use committee guidelines.

Reagents

The high m.w. dsRNA (long poly I:C [LPIC], catalog: tlrl-pic-5), low m.w. dsRNA (short poly I:C, catalog: tlrl-picw), R848 (TLR7/8 ligand, catalog: tlrl-r848), TSLP (thymic stromal lymphopoietin), α-CD40 (neutralizing mAb against human CD40L, catalog: mabg-h40l-3), LPS (TLR4 ligand, catalog: tlrl-pglps), CpG A (TLR9 agonist, catalog: tlrl-2216), and CpG B (TLR9 agonist, catalog: tlrl-2006) were from InvivoGen. Lipofectamine 3000 was from Invitrogen. The proteasome inhibitor MG132 was from Sigma-Aldrich. The following Abs were used for immunoprecipitation: anti-MAVS (1:100) (4983S; Cell Signaling Technology) and anti-TRIM29 (1:100) (A301-210A; Bethyl). The following Abs were used for immunoblot analysis: anti-MAVS (1:1000) (4983S; Cell Signaling Technology), anti-TRIM29 (1:1000) (A301-210A; Bethyl), anti-TRIM29 (1:1000) (sc-33151; H-300; Santa Cruz Biotechnology), anti-ubiquitin (1:1000) (sc-8017; Santa Cruz), K11-specific anti-ubiquitin (1:1000) (MABS107-I; Millipore-Sigma), anti-GAPDH (1:10,000) (clone GAPDH-71.1, G9295; Sigma-Aldrich), anti-hemagglutinin (HA) (1:5000) (clone HA-7, H6533; Sigma-Aldrich), anti–β-actin (1:20,000) (clone AC-15, A3854; Sigma-Aldrich), and anti-Myc (1:5000) (ab1326; Abcam). Anti-HA and anti-Myc beads were from Sigma-Aldrich. Lentiviral vectors for short hairpin RNA (shRNA) were as follows (all from Open BioSystems): human TRIM29 (clone TRCN0000016348 [TRIM29-1]) and clone TRCN0000016352 [TRIM29-2]). The IFN-β and IFN-α ELISA kits were from PBL InterferonSource. The Dual-Luciferase Reporter Assay System (E1910) was from Promega.

Cells culture and lentiviral infection

Human mDCs were isolated from buffy coats of individual healthy donors and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Invitrogen; Life Technologies) as previously described (26, 27). HEK293T cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Invitrogen; Life Technologies). All cell lines were tested without mycoplasma contamination. The mDCs were infected with a pLKO.1 lentiviral vector carrying a scrambled shRNA (RHS6848; Open BioSystems) or target gene sequences (Open BioSystems) as described in our previous studies (20, 25, 28, 29). After 24 h of culture, cells were selected by the addition of puromycin (2 ng/ml) to the medium. Cells were stimulated for 16 h with LPIC (20 μg/ml) delivered by Lipofectamine 3000. The knockdown efficiency was detected with immunoblot analysis.

Virus infection

For reovirus infection in mice, 4- to 5-wk-old mice were inoculated i.p. with 2 × 107 PFU of reovirus (reovirus type 3 Dearing [T3D] strain) in PBS. Reovirus T3D strain was purchased from American Type Culture Collection (VR-824). Mice were euthanized at various time points following infection, and tissues were collected for analysis.

For analysis of reovirus replication, mice were euthanized at defined intervals postinoculation, and organs (heart, liver, spleen, brain, and intestine) were excised into 1 ml of PBS and homogenized by freezing, thawing, and sonication. Intestines were transected proximally at the gastroduodenal junction and distally at the rectum before homogenization in 1 ml of PBS.

Virus plaque titration

For reovirus infection in mice, viral titers in organ homogenates from infected mice were determined by plaque assay on L929 cells (30). Weights of organs were measured before the assay, and PFU were calculated per milligrams of tissue. Briefly, tissue was homogenized in 800 μl of PBS. The homogenates were treated with chloroform (10% final concentration) and centrifuged briefly, and serial dilutions of the aqueous supernatants were incubated on L929 cells at room temperature. After 1 h, the inoculum was removed, and cells were covered with 2% agar solution with amphotericin-B. After 6 d, 2% agar solution containing 2% neutral red solution was added, and plaques were visualized with neutral red on the second day.

In vitro pull-down and immunoblot analysis

For the preparation of purified MAVS and TRIM29, HEK293T cells were transfected with expression plasmids encoding full-length or truncated versions of HA- or Myc-tagged MAVS or TRIM29. Lysates were prepared from the transfected cells, followed by incubation with anti-HA or anti-Myc beads. Proteins were eluted from the beads after beads were washed six times with PBS. For precipitation with anti-HA or anti-Myc beads, purified Myc-tagged WT MAVS or truncations of MAVS were incubated for 2 h with purified HA-tagged TRIM29 or purified HA-tagged TRIM29, or truncations of TRIM29 were incubated for 2 h with purified Myc-tagged MAVS. Beads were added, and after 2 h of incubation, the bound complexes were pelleted by centrifugation. Proteins and beads were analyzed by immunoblot analysis with anti-HA or anti-Myc Abs (20, 26, 27, 31–33).

Ubiquitination

HEK293T cells were transfected with an expression plasmid encoding Myc-tagged full-length MAVS and with or without coexpression of HA-tagged full-length TRIM29 and its mutant T29-N. At 24 h after transfection, cells were treated with 25 μM MG132 for 3 h and then were collected for analysis (20, 25, 29, 34). Briefly, cells were lysed, and the cell lysis was heated to 65°C for 5 min. After spinning down, the supernatants were collected, immunoprecipitated with anti-Myc beads, and then analyzed by immunoblot.

Confocal microscopy

BMDCs isolated from the WT mice were infected with reovirus for 4 h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, blocked for 30 min with 5% BSA, incubated with rabbit anti-MAVS polyclonal Ab (catalog no.: 14341-1-AP; Proteintech) and mouse anti-TRIM29 mAb (sc-166707; Santa Cruz) for 2 h followed by Alexa Fluor 594 goat anti-rabbit secondary Ab and Alexa Fluor 488 goat anti-mouse secondary Ab for 1 h, and examined with confocal microscopy. Images of zoomed single cells were quantified with Nikon Confocal Software.

Quantitative RT-PCR

RNA was isolated using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. The isolated RNA was used to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). iTaq SYBR Green Supermix with ROX (Bio-Rad) was used for quantitative RT-PCR. PCRs were performed in triplicate. The following primers were used: IFN-β1 forward, 5′-CCCTATGGAGATGACGGAGA-3′ and reverse, 5′-TCCCACGTCAATCTTTCCTC-3′; IFN-α5 forward, 5′-AGTGAGCTGACCCAGCAGAT-3′ and reverse, 5′-CAGGGGCTGTGTTTCTTCTC-3′; IL-6 forward, 5′-CTGATGCTGGTGACAACCAC-3′ and reverse, 5′-TTCTGCAAGTGCATCATCGT-3′; and GAPDH forward, 5′-AACTTTGGCATTGTGGAAGG-3′ and reverse, 5′-ACACATTGGGGGTAGGAACA-3′.

Luciferase reporter gene assay

Human HEK293T cells were seeded on 48-well plates (1 × 105 cells per well), then transfected with reporter vectors for Ifnb-firefly luciferase (100 ng) or nfκb-firefly luciferase and Renilla luciferase (1 ng) plus expression vector for WT MAVS or MAVS mutants (100 ng) with or without expression vector for TRIM29 (300 ng). Empty control vector was added so that a total of 550 ng of vector DNA was transfected into each well of cells. At 24 h after transfection, cells were collected for luciferase activity analysis. Luciferase activity in total cell lysates was detected by Dual-Luciferase Reporter Assay (20).

Statistical analysis

A two-tailed unpaired Student t test was used for statistical analysis with Microsoft Excel and GraphPad Prism Software. The p values <0.05 were considered significant unless specifically indicated.

Results

TRIM29 is involved in type I IFN production by human mDCs in response to poly I:C

To investigate the function of TRIM29 in humans, we started with analyses of TRIM29 expression in human immune cells. Microarray gene expression analysis showed that TRIM29 was specifically expressed in poly I:C–induced human mDCs (data not shown). To further confirm the unique expression of TRIM29 in mDCs, human primary mDCs were isolated from PBMCs of one random donor and stimulated with R848 (TLR7/8 ligand, tlrl-r848; InvivoGen), poly I:C (high m.w. LPIC, RLR ligand), TSLP, α-CD40 (neutralizing mAb against human CD40L, mabg-h40l-3; InvivoGen), LPS (TLR4 ligand), CpG A (TLR9 agonist), and CpG B (TLR9 agonist). The real-time PCR analysis showed that TRIM29 was indeed uniquely expressed in human mDCs stimulated by poly I:C but not by other stimulants (Fig. 1A). To further investigate the biological function of poly I:C highly induced TRIM29 in mDCs, we first established stable knockdown of TRIM29 mDC cell lines through the use of shRNA. Two distinct TRIM29-targeting shRNAs (Trim29 no. 1 and Trim29 no. 2) produced efficient knockdown of TRIM29 expression (Fig. 1B). Additionally, MAVS-targeting shRNA efficiently knocked down the MAVS expression (Fig. 1B). These cells were then stimulated by poly I:C, and the production of IFN-β by the cultured cells was measured by ELISA. Consistent with published data, knockdown of MAVS abrogated the production of IFN-β in mDCs that had been induced by cytosolic poly I:C. In contrast, knockdown of TRIM29 enhanced the IFN-β production in mDCs up to 3-fold in response to intracellular dsRNA (Fig. 1C). These data suggested that TRIM29 negatively regulated type I IFN production in dendritic cells in response to dsRNA.

FIGURE 1.
FIGURE 1.

The induced TRIM29 plays an important role in producing type I IFN by human mDCs in response to poly I:C. (A) Human mDCs were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells induced or not to real-time PCR. The profile of TRIM29 expression in different cells is indicated. The relative expression of TRIM29 was compared by plotting the values extracted from the gene expression database. A value <1 indicated the absence of gene expression. (B) Immunoblot analysis of TRIM29 and MABVS in human mDCs treated with control shRNA with a scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: Trim29 no. 1 and Trim29 no. 2), or shRNA targeting mRNA encoding MAVS. GAPDH serves as a loading control throughout. (C) ELISA of IFN-β in human primary mDCs treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16 h with LPIC (20 μg/ml) delivered by Lipofectamine 3000. Data are representative of three independent experiments with similar results (mean + SD). **p < 0.01, ***p < 0.001, unpaired t test. N-STM, cells without stimulation.

TRIM29 KO enhanced the production of type I IFN and IL-6 in BMDCs and BMDMs in response to poly I:C

To further determine the function of TRIM29 in primary mice cells, we prepared BMDCs and BMDMs from WT mice and Trim29-deficient mice. We then stimulated those BMDCs or BMDMs for 16 h with poly I:C. As a result, Trim29-deficient BMDCs produced 2- to 3-fold more type I IFN, including IFN-α (Fig. 2A), IFN-β (Fig. 2B), and IL-6 (Fig. 2C), in response to poly I:C than did WT BMDCs. Similarly, compared with WT BMDMs, Trim29-KO BMDMs produced up to 3-fold more IFN-α (Fig. 2D), IFN-β (Fig. 2E), and IL-6 (Fig. 2F) in response to poly I:C. Additionally, the quantitative PCR data confirmed that Trim29-deficient BMDCs or BMDMs produced more IFN-α5, IFN-β1, and IL-6 in mRNA levels in response to poly I:C than did WT BMDCs (Supplemental Fig. 1A) or BMDMs (Supplemental Fig. 1B).These data indicated a negative role of TRIM29 in murine BMDCs and BMDMs in response to poly I:C.

FIGURE 2.
FIGURE 2.

KO-TRIM29 enhanced the production of type I IFN and IL-6 in BMDCs and BMDMs in response to poly I:C. (A–F) ELISA of IFN-α (A and D), IFN-β (B and E), and IL-6 (C and F) in BMDCs (A–C) or BMDMs (D–F) from WT and Trim29-KO mice after 16 h of stimulation with LPIC (20 μg/ml) or short poly I:C (SPIC, 20 μg/ml) delivered by Lipofectamine 3000. Data are representative of three independent experiments with similar results (mean + SD). *p < 0.01, **p < 0.001, unpaired t test. Mock, cells without stimulation.

TRIM29 negatively regulates type I IFN production in BMDCs and BMDMs upon RNA virus infection

The RLRs are cytosolic PRR proteins recognizing viral RNA species, including dsRNA and 5′-triphosphate RNA (35). In the case of RNA viruses, viral RNA replicase generates 5′-triphosphate RNA and/or dsRNA in ample amounts during replication and transcription of viral RNA genomes. The dsRNA is produced during infection by dsRNA virus reovirus. So, we employed the dsRNA virus T3D strain to investigate the role of TRIM29 in sensing RNA virus. We then prepared BMDCs and BMDMs from WT mice and Trim29-deficient mice and infected those BMDCs and BMDMs for 12 or 20 h with reovirus. Trim29-deficient BMDCs or BMDMs produced 2- to 4-fold more IFN-β and IFN-α in response to reovirus than did WT BMDCs or BMDMs (Fig. 3A, 3B). The quantitative PCR data also confirmed that Trim29-deficient BMDCs or BMDMs produced more IFN-α5 and IFN-β1 in mRNA levels in response to reovirus infection than did WT BMDCs (Supplemental Fig. 2A) or BMDMs (Supplemental Fig. 2B). Additionally, there is much less reovirus production in Trim29-deficient BMDCs (Fig. 3C) or BMDMs (Fig. 3D) than in WT BMDCs or BMDMs after reovirus infection. These data demonstrated that TRIM29 played a negative role in murine BMDCs and BMDMs in sensing RNA virus.

FIGURE 3.
FIGURE 3.

TRIM29 negatively regulates type I IFN production in BMDCs and BMDMs upon RNA virus infection. (A and B) ELISA of IFN-α and IFN-β in BMDCs (A) or BMDMs (B) from WT and Trim29-KO (KO) mice mock-infected or infected with reovirus at a multiplicity of infection of 5 for 12 or 20 h. (C and D) The viral titers of the supernatants from BMDCs (C) or BMDMs (D) from WT and Trim29-KO (KO) mice mock-infected or infected with reovirus at a multiplicity of infection of 5 by plaque assay. Mock, cells without infection. Data are representative of three independent experiments with similar results (mean + SD). **p < 0.01, ***p < 0.001, unpaired t test.

TRIM29 plays an important role in host defense in vivo

We next evaluated the importance of TRIM29 in host defense against RNA viral infection in vivo. We first challenged WT mice and Trim29-KO mice i.p. with T3D strain reovirus and monitored survival over time. The challenge of WT mice with reovirus led to lethal infection (Fig. 4A). In contrast, Trim29-KO mice had significantly higher survival rates after reovirus infection (Fig. 4A). Next, we infected WT mice and Trim29-deficient mice i.p. with reovirus and then measured IFN-β in heart homogenates from infected mice. As expected, Trim29-deficient mice produced 2- to 3-fold more IFN-β than did WT mice in response to reovirus (Fig. 4B). Additionally, we harvested intestine, spleen, liver, heart, and brain at days 2 and 4 postinfection and determined viral titers of reovirus in those organs by plaque assay. We detected significantly less reovirus in Trim29-deficient mice than in WT mice, especially at day 4 postinfection (Fig. 4C). These data indicated that RNA virus hijacked TRIM29 to shut down the innate immune responses to immune evasion and viral pathogenesis.

FIGURE 4.
FIGURE 4.

TRIM29 plays an important role in host defense against RNA virus infection in vivo. (A) Survival of WT and Trim29-KO mice (n = 8 per strain) after i.p. injection of reovirus (2 × 107 PFU per mouse). (B) The WT (TRIM29+/+) and Trim29-deficient (TRIM29−/−) mice were inoculated i.p. with 1 × 107 PFU of reovirus T3D strain. At days 2 and 4 postinoculation, mice were euthanized and hearts were excised, frozen at −80°C, thawed, and homogenized in PBS. Levels of IFN-β in heart homogenates were quantified by ELISA. (C) The WT (WT+/+) and Trim29-deficient (KO−/−) mice were inoculated i.p. with 1 × 107 PFU of reovirus. At day 2 and day 4 postinoculation, mice were euthanized; intestine, spleen, liver, heart, and brain were excised; and viral titers in organ homogenates were determined by plaque assay. Results are expressed as mean viral titers for three animals for each time point. Error bars indicate SEs of the mean. Data are representative of three independent experiments with similar results (mean + SD). ***p < 0.001, unpaired t test. Mock, mice without infection.

TRIM29 binds directly to MAVS in RNA-sensing signaling pathway

To further investigate how TRIM29 regulates in RNA virus–induced signaling events, we identified TRIM29-interacting proteins by immunoprecipitation with an Ab against TRIM29 (anti-TRIM29) in the mouse mDC line D2SC, followed by protein sequencing by liquid chromatography–mass spectrometry. We identified the MAVS among the group of TRIM29-interacting proteins. We next determined the possible interaction between TRIM29 and MAVS in BMDCs at the endogenous protein level. Anti-MAVS Ab or anti-TRIM29 Ab, but not control IgG, could pull down TRIM29 and MAVS, separately, in BMDCs induced by dsRNA stimulation. By contrast, Anti-MAVS Ab or anti-TRIM29 Ab could not pull down TRIM29 or MAVS without dsRNA stimulation, suggesting there was indeed interaction between TRIM29 and MAVS in BMDCs under the condition of cytosolic dsRNA stimulation (Fig. 5A). To further map the binding sites between MAVS and TRIM29, we analyzed the interactions between HA-tagged recombinant TRIM29 and Myc-tagged recombinant full-length MAVS and truncation mutants of MAVS. Both the recombinant full-length and the C-terminal transmembrane domains of MAVS, but not its N-terminal CARD and Proline domains, bound TRIM29 (Fig. 5B), suggesting that the C-terminal transmembrane domain of MAVS interacted with TRIM29 (Fig. 5B). Next, we analyzed the interactions between Myc-tagged MAVS together with HA-tagged full-length TRIM29 and truncation mutants of TRIM29. As a result, the full-length TRIM29 and all mutants except its N-terminal domain could interact with MAVS (Fig. 5C), suggesting that the C-terminal domain of TRIM29 bound MAVS (Fig. 5C). By using a luciferase reporter assay of the IFN-β promoter established in human HEK293T cells, we found that overexpression of MAVS activated the IFN-β promoter activity (Fig. 5D). However, overexpression of WT TRIM29 (T29-a) and its truncate containing the interaction domain with MAVS (T29-b) inhibited the activation of the IFN-β promoter induced by MAVS (Fig. 5D), whereas overexpression of TRIM29 truncate without the interaction domain with MAVS lost its inhibitory effect (Fig. 5D). Furthermore, immunofluorescence of TRIM29 and MAVS showed that endogenous TRIM29 could colocalize with endogenous MAVS in the cytosol in BMDCs after reovirus infection (Fig. 5E). Collectively, these data suggested that TRIM29 could bind directly to key adaptor MAVS in the RNA virus sensing signaling pathway.

FIGURE 5.
FIGURE 5.

TRIM29 binds directly to MAVS. (A) Immunoblot analysis of endogenous proteins of TRIM29 and MAVS precipitated with anti-MAVS, anti-TRIM29, or control IgG from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with poly I:C (25 μg/ml) for 8 h and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. (B) Full-length MAVS and serial truncations of MAVS with deletion of various domains (top). Below, Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA (bottom blot) and immunoblot analysis of purified Myc-tagged full-length MAVS and MAVS truncation mutants alone with anti-Myc (top blot) or after incubation with HA-tagged full-length TRIM29 and immunoprecipitation with anti-HA (middle blot). (C) Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains (left margin); numbers at ends indicate amino acid positions (top). Below, Immunoblot analysis of purified Myc-tagged MAVS with anti-Myc (bottom blot) and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone (top blot) or after incubation with Myc-tagged MAVS and immunoprecipitation with anti-Myc (middle blot). (D) Activation of the IFN-β promoter in human HEK293T cells transfected with an IFN-β promoter luciferase reporter plus expression vector (each 100 ng) for WT MAVS or expression vector for WT TRIM29 (T29-a) or TRIM29 mutants T29-b and T29-g; results are presented relative to those of Renilla luciferase (cotransfected as an internal control). (E) Colocalization of endogenous TRIM29 and MAVS in BMDCs. Confocal microscopy of BMDCs infected with reovirus for 4 h. MAVS was stained with rabbit anti-MAVS polyclonal Ab (catalog no.: 14341-1-AP; Proteintech) followed by Alexa Fluor 594 goat anti-rabbit secondary Ab (green), whereas TRIM29 was stained with mouse anti-TRIM29 mAb (sc-166707; Santa Cruz Biotechnology) followed by Alexa Fluor 488 goat anti-mouse secondary Ab (red). DAPI served as the nuclei marker (blue). Scale bars, 20 μm. Data are representative of three independent experiments.

TRIM29 ubiquitinates and degrades MAVS

Because TRIM29 is an E3 ubiquitin ligase, we next investigated whether MAVS is the ubiquitination target of TRIM29. We then coexpressed HA-TRIM29 with Myc-MAVS or vector control in HEK293T cells with or without treatment of MG132 and analyzed the expression of MAVS and TRIM29. As a result, TRIM29 could significantly degrade the target protein MAVS, compared with the vector control (Fig. 6A). Additionally, the treatment of MG132 could rescue the expression of MAVS (Fig. 6A), suggesting that MAVS is indeed the ubiquitination target of TRIM29. To determine whether the expression of TRIM29 was regulated by poly I:C stimulation, we stimulated the WT and TRIM29 KO BMDCs with poly I:C and then measured the expression of TRIM29 and MAVS in BMDCs after poly I:C stimulation. TRIM29 was upregulated in BMDCs after poly I:C stimulation, whereas MAVS was downregulated after poly I:C stimulation owing to the degradation by TRIM29 (Fig. 6B). Furthermore, the IRF3 and p65 were activated much more strongly in TRIM29 KO BMDCs than those in WT BMDCs after poly I:C stimulation (Fig. 6B). These data indicated that TRIM29 was highly inducible and degraded MAVS much more strongly after poly I:C stimulation.

FIGURE 6.
FIGURE 6.

TRIM29 induces ubiquitination and degradation of MAVS by K11 linkage. (A) Immunoblot analysis of Myc-tagged MAVS (top blot), HA-tagged TRIM29 (middle blot), and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for Myc-tagged MAVS and empty vector or expression vector for HA-tagged TRIM29, with or without treatment of 5 μM MG132. (B) Immunoblot analysis of TRIM29 (top blot), MAVS (second blot), phosphorylated IRF3 (third blot), phosphorylated p65 (fourth blot), and β-actin (bottom blot) in WT and TRIM29 KO BMDCs stimulated for various times (above lanes) with poly I:C (10 μg/ml). (C) Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot), and K11-linked ubiquitination (third blot) of Myc-tagged MAVS in HEK293T cells transfected with empty vector or expression vector for HA-tagged TRIM29, truncated T29-g (losing binding site of MAVS), and stimulated for 4 h with poly I:C (20 μg/ml), assessed after immunoprecipitation with anti-Myc. Immunoblot analysis whole-cell lysates with anti-HA (fifth blot) and anti–β-actin (bottom). (D) Immunoblot analysis of TRIM29 in WT and KO BMDCs (top) and of the abundance (second blot), total ubiquitination (third blot), and K11-mediated ubiquitination (bottom blot) of MAVS in those cells, stimulated for 4 h with poly I:C (10 μg/ml) and assessed after immunoprecipitation with anti-MAVS. Data are representative of three experiments.

To investigate whether the ubiquitination of MAVS was dependent on the binding site of TRIM29 with MAVS, we transfected the HEK293T cells to coexpress Myc-tagged MAVS and HA-tagged full-length TRIM29, as truncated TRIM29 lacked the binding site of MAVS (T29-g). We stimulated the cells for 6 h with dsRNA and treated the cells with MG132 for 6 h, then prepared cell lysates and incubated them for 5 min at 90°C with 1% SDS to disrupt protein–protein interactions, which was followed by immunoprecipitation of Myc-tagged MAVS. Immunoblot analysis of HA or ubiquitin demonstrated that the ubiquitination of MAVS was strongly enhanced by overexpression of TRIM29 but not by overexpression of T29-g (Fig. 6C). Immunoblot analysis of K11-linked ubiquitin further demonstrated that TRIM29 induced ubiquitination of MAVS by K11-mediated linkage (Fig. 6C). To determine whether TRIM29 was responsible for the ubiquitination of MAVS ex vivo, we stimulated BMDCs from WT mice or Trim29-KO mice for 4 h with poly I:C. Cell lysates were prepared and analyzed for the ubiquitination of MAVS. MAVS was modified via K11-mediated ubiquitination in BMDCs from WT mice but not Trim29-KO mice (Fig. 6D). Together, these data indicated that TRIM29 targeted MAVS and induced its ubiquitination for protein degradation by K11 linkage.

Lys371, Lys420, and Lys500 of MAVS are necessary for TRIM29-mediated signaling

To determine the MAVS ubiquitination sites, we replaced each of these lysine residues individually with arginine (K371R, K420R, K461R, K500R, and triple mutation K371RK420RK500R). We coexpressed HA-tagged TRIM29 with Myc-tagged WT MAVS and its mutants, noted above, in HEK293T cells and detected their expressions. As a result, TRIM29 was expressed similarly. Moreover, TRIM29 could strongly degrade the WT MAVS and its mutants (K461R), whereas the MAVS mutants K371R, K420R, and K500R could be partially degraded by TRIM29. In contrast, TRIM29 could not degrade the triple mutant MAVS (K371RK420RK500R) (Fig. 7A), suggesting the Lys371, Lys420, and Lys500 of MAVS are the ubiquitination sites mediated by TRIM29. To further determine whether the ubiquitination of MAVS by TRIM29 was affected by the mutation of MAVS, we transfected the HEK293T cells to express Myc-tagged MAVS or its mutants noted above and HA-tagged full-length TRIM29 with the treatment of MG132. Cells were stimulated for 4 h with dsRNA. Cell lysates were prepared and followed by immunoprecipitation of Myc-tagged MAVS. Immunoblot analysis demonstrated that both Myc-tagged MAVS or its mutants and HA-tagged full-length TRIM29 were expressed similarly. As expected, the ubiquitination of MAVS or its mutant K461R was strongly enhanced by overexpression of TRIM29 (Fig. 7B). However, the ubiquitination of MAVS mutant K371R, K420R, or K500R by TRIM29 was sharply reduced (Fig. 7B). Especially, ubiquitination of MAVS triple mutant (K371RK420RK500R) was completely lost (Fig. 7B).

FIGURE 7.
FIGURE 7.

Lys371, Lys420, and Lys500 of MAVS are necessary for TRIM29-mediated signaling. (A) Immunoblot analysis of Myc-tagged MAVS and its mutations (top blot), HA-tagged TRIM29 (middle blot), and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for HA-tagged TRIM29 and expression vectors for Myc-tagged WT full-length MAVS and its mutations. (B) Immunoblot analysis of HA-tagged TRIM29 (top blot), Myc-tagged MAVS and its mutations (second blot), β-actin (third blot), and K11-linked ubiquitination (bottom blot) in HEK293T cells cotransfected with expression vector for Myc-tagged MAVS or its mutations (above lanes) and expression vector for HA-tagged TRIM29 with treatment of 5 μM MG132 (above lanes). (C and D) Activation of the IFN-β promoter (C) or NF-κB promoter (D) in human HEK293T cells transfected with an IFN-β promoter luciferase reporter (C) or NF-κB promoter luciferase reporter (D), plus expression vector (each 100 ng) for WT MAVS or various MAVS mutants alone or expression vector for TRIM29 plus WT MAVS or MAVS mutants; results are presented relative to those of Renilla luciferase (cotransfected as an internal control). Data are representative of three independent experiments with similar results (mean + SD).

Through the use of a luciferase reporter assay of the IFN-β promoter and NF-κB promoter, established in the human HEK293T cells, we found that overexpression of WT MAVS enhanced the activity of the IFN-β promoter (Fig. 7C) and NF-κB promoter (Fig. 7D), Overexpression of mutant MAVS with the K371R, K420R, K461R, K500R, and K371RK420RK500R substitution could still induce the activation of the IFN-β promoter (Fig. 7C) and NF-κB promoter (Fig. 7D). In addition, overexpression of TRIM29 inhibited the activation of the IFN-β promoter and NF-κB promoter in HEK293T cells expressing WT MAVS or its mutant with the K461R substitution (Fig. 7C, 7D). However, overexpression of TRIM29 did not inhibit the activity of the IFN-β promoter or NF-κB promoter in HEK293T cells expressing mutant MAVS with K371R, K420R, K500R, or K371RK420RK500R substitution (Fig. 7C, 7D). These data indicated that Lys371, Lys420, and Lys500 were essential for TRIM29-mediated ubiquitination and regulation of MAVS.

Discussion

Immune protection against viral infections relies on activation of the innate immune response to restrict viral replications. Activation of innate immune cells is tightly regulated, in that robust antiviral immunity is achieved without triggering excessive autoimmune inflammation. Previously, we found that TRIM29 was constitutively highly expressed in alveolar macrophages and controlled the local innate immunity in the respiratory tract (20). Recently, we also found that TRIM29 promoted DNA virus infections by inhibiting the innate immune response through degrading STING (25). However, the precise mechanisms of how antiviral signaling activation is strictly controlled during RNA virus infections still remain incompletely defined. In this study, we found the E3 ligase TRIM29 was specifically expressed in poly I:C–activated human mDCs. TRIM29 was highly induced in human mDCs in response to poly I:C and played a negative role in type I IFN production. The deficiency in TRIM29 resulted in enhanced type I IFN responses to poly I:C and RNA virus infection. Importantly, the challenge of WT mice with reovirus led to lethal infection. In contrast, deletion of TRIM29 protected the mice from developing lethal complications. Mechanistically, TRIM29 was shown to interact with MAVS, the key adaptor in the RNA-sensing pathway. Importantly, TRIM29 induced ubiquitination and degradation of MAVS through K11-linked polyubiquitination. These data suggest that RNA virus hijacks the E3 ligase TRIM29 to shut down the host innate immune response for immune evasion.

Similar to many pathways, RLR signaling is tightly regulated to achieve an orchestrated response aimed at maximizing antiviral immunity and minimizing immune- or nonimmune-mediated collateral damage. To achieve an appropriately balanced response, downregulation of antiviral signaling is equally important to its activation. Polyubiquitination has been reported to regulate the activation of MAVS (36). Several E3 ligases have been identified that modulate the polyubiquitination of MAVS, including AIP4 (16, 37), RNF5 (38), RNF125 (39), Smurf1 and Smurf2 (40, 41), TRIM25 (42), and MARCH 5 (43). However, all of these E3 ligases promote K48-linked ubiquitination of MAVS. Recently, TRIM31 (44) and kinases IKK (45) were identified to mediate positive modification of MAVS through the K63-linked polyubiquitination and phosphorylation, respectively. In our study, we first identified TRIM29 as an E3 ligase for the K11-linked polyubiquitination of MAVS. To our knowledge, TRIM29 is the first E3 ligase identified in negatively regulating the activation of MAVS through its ubiquitination in the K11 linkage.

So far, a large number of molecules were proposed and identified as important regulators in the antiviral signaling pathway of type I IFN production. But very few concepts were investigated and verified in vivo using KO mice. In the current study, our observations based on the study of Trim29−/− mice solidify a role for TRIM29 as an in vivo checkpoint of type I IFN response. We have extended these findings to the RNA virus and identified TRIM29 as a key negative regulator in the host defense against the RNA virus.

Type I IFNs play a critical role in eliminating not only the invading pathogens but also many autoimmune diseases via various immune modulatory actions (46). Recent onset of type 1 diabetes is strongly correlated with infection by RNA viruses such as enteroviruses (47, 48). In this context, the identification of TRIM29 as a negative regulator of key adaptor MAVS in the RNA-sensing signaling pathway will have important implications for the understanding of not only antiviral innate immunity but also the pathogenesis of human autoimmune diseases.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Wellcome Trust Sanger Institute Mouse Genetics Project and its funders for providing the mutant mouse line Trim29 and the European Mouse Mutant Archive (http://www.emmanet.org) partner from which the mouse line was received.

Footnotes

  • This work was supported by Lupus Research Alliance Grant 519418 (to Z.Z.) and National Institutes of Health Grant R01AI080779 (to X.C.L.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BMDC
    bone marrow–derived dendritic cell
    BMDM
    bone marrow–derived macrophage
    HA
    hemagglutinin
    KO
    knockout
    LPIC
    long poly I:C
    MDA5
    melanoma differentiation–associated protein 5
    mDC
    myeloid dendritic cell
    RIG-I
    retinoic acid–inducible gene I
    RLR
    RIG-I–like receptor
    shRNA
    short hairpin RNA
    T3D
    type 3 Dearing
    TRIM
    tripartite motif protein
    WT
    wild-type.

  • Received November 15, 2017.
  • Accepted April 30, 2018.

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