USP20 Promotes Cellular Antiviral Responses via Deconjugating K48-Linked Ubiquitination of MITA

The innate immune system deploys pattern-recognition receptors (PRRs) to detect structurally conserved pathogen-associated molecular patterns of invading pathogens, which constitutes the first line of host defense against infectious microbes (1). Nucleic acids including RNA, DNA, and RNA–DNA hybrid are classical pathogen-associated molecular patterns recognized by PRRs and trigger a series of signaling cascades (2, 3). Among the identified PRRs, TLRs such as TLR3 and TLR7/8 and retinoic acid–inducible gene I (RIG-I)-like receptors (RLRs) including RIG-I and melanoma differentiation-associated gene 5 (MDA5) detect endosomal or cytoplasmic RNA, respectively. In contrast, TLR9 and a number of DNA sensors including RNA polymerase III, IFN-γ–inducible protein 16 (IFI16), DEAD-box helicase 41 (DDX41), and DNA-dependent activator of IRFs (DAI) have been reported to detect endosomal or cytoplasmic DNA in location-, sequence- and/or cell type–dependent manner. Particularly, the nucleotidyl transferase cyclic GMP-AMP (cGAMP) synthase detects dsDNA, ssDNA, and DNA–RNA hybrid independent of the sequences and is recognized as a “universal” cytoplasmic DNA sensor in various cell types (4, 5). Upon binding to cytoplasmic DNA, cGAMP synthase catalyzes the synthesis of cGAMP, which serves as a second messenger binding to the endoplasmic reticulum and mitochondrial adaptor protein mediator of IRF3 activation ([MITA] also known as STING, MPYS, and ERIS) and induces dimerization or oligomerization of MITA (6–10). MITA further recruits adaptor proteins such as TRAF3 and TRAF6 and kinases TBK1/IKKε and IKKα/β/γ complex to activate transcription factors IRF3/7 and NF-κB to induce expression of a large array of downstream genes.

Studies with MITA-deficient mice demonstrate that MITA is the sole adaptor protein downstream of the cytoplasmic DNA sensors (11). Therefore, the activity of MITA must be strictly regulated to elicit protective immune responses and avoid excessive autoimmune responses. Posttranslational modifications (PTMs) play essential roles in regulating the activity and fate of target proteins, and multiple PTMs have been reported to regulate the activity of MITA (3). For example, TBK1 and ULK1 phosphorylate MITA at Ser366, which promotes the recruitment of IRF3 and the degradation of MITA, respectively (12, 13). Recently, it has been reported that TRIM38 and SENP2 target MITA for sumoylation and desumoylation at the early and late phase of HSV-1 infection, respectively (14). Such a reversible PTM modulates the degradation of MITA through the chaperone-mediated autophagy pathway without affecting the ubiquitination of MITA. The E3 ubiquitin ligases TRIM56, TRIM32, and MUL1 target MITA for K63-linked ubiquitination, and AMFR promotes K27-linked ubiquitination of MITA upon viral infection, which promotes the recruitment of TBK1 to MITA and the induction of type I IFNs and proinflammatory cytokines (15–18). However, genetic studies show that deletion of TRIM56 or TRIM32 has minimal effect on ubiquitination of MITA in mouse cells (16, 19). In contrast, TRIM29 and RNF5 target MITA for K48-linked ubiquitination and proteasomal degradation and downregulates antiviral immune response (20–22). As the reverse process of ubiquitination, however, the deubiquitinating process of MITA and the physiological relevance have not been fully investigated.

Deubiquitination is mediated by deubiquitinating (DUB) enzymes that play diverse roles in various physiological or pathological processes (23, 24). We have previously reported that USP13 constitutively interacts with MITA and disassociates from MITA after HSV-1 infection to keep K27-linked ubiquitination of MITA in check, thereby turning down excessive immune responses against HSV-1, whereas USP18 interacts with MITA after viral infection and recruits USP20 to deconjugate K48-linked polyubiquitin chains from and prevent proteasomal degradation of MITA (25, 26). Although it has been reported that USP20 targets various proteins including ULK1 (27), Rad17 (28), β adrenergic receptor (29), TRAF6 (30), Claspin (31, 32), and β-catenin (33) for deubiquitination and thereby regulates IL-1β signaling, DNA damage repair, tumorigenesis, and chemo-resistance of cancers, the genetic evidence of such a regulation is still lacking.

In this study, we have generated USP20-deficient mice and examined the role of USP20 in innate antiviral signaling. We found that knockout of USP20 impairs DNA virus-triggered activation of IRF3 and NF-κB and expression of downstream genes and promotes K48-linked ubiquitination and proteasomal degradation of MITA after HSV-1 infection. Consistently, USP20 deficiency leads to potentiated HSV-1 replication in cells and in vivo and USP20-deficient mice exhibit increased susceptibility to lethal HSV-1 infection. Our findings have provided genetic evidence that USP20 plays an essential role in cellular antiviral responses by targeting MITA for deubiquitination.

The Usp20^+/− mice were generated by Beijing Vitalstar Biotechnology Company through CRISPR/Casp9–mediated gene editing. In brief, Cas9 mRNA and guide RNA (5′-GACCTTCGCCAGTGTACCTGTGG-3′ and 5′-GTCCTACTGTGCAGCACTCGTGG-3′) were in vitro transcribed and injected into the fertilized eggs that were transplanted into pseudopregnant mice. The tail DNA of F0 mice was amplified with PCR sequence, and the chimeras were crossed with wild-type C57BL/6 mice to obtain the Usp20^+/− mice. The F1 Usp20^+/− mice were further crossed with wild-type C57BL/6 mice for at least three generations. The genotyping of the Usp20^−/− mice was confirmed by sequencing of the PCR fragments amplified from the genomic DNA isolated from tails using the following primers: Usp20^+/+ and Usp20^−/− forward 5′-TGGGACAAGGACAAGAGCAGG-3′; Usp20^+/+ reverse 5′-CCCATAGGTTAGGTCCAGCAAC-3′; Usp20^−/− reverse 5′-GCAGTGTGTTTATTTAACTTCACGGTA-3′.

The age- and sex-matched Usp20^+/+ and Usp20^−/− littermates were randomized into groups for animal studies. All mice were housed in the specific pathogen-free animal facility at Wuhan University and all animal experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of Wuhan University.

The IFN-stimulating DNA (ISD)45, HSV60, DNA90, HSV120, and poly(I:C) were described previously (34). MG132 and 3MA were purchased from Sigma-Aldrich. IFN-α was from BioLegend (75802). Rabbit Ab for USP20 (A301-189A-T-2) was from Abcam, Ab for K48-specific anti-ubiquitin (05-1307) was from Millipore, HRP-conjugated goat anti-mouse or rabbit IgG (PA1-86717 and SA1-9510) was obtained from Thermo Fisher Scientific, HRP-conjugated mouse anti-FLAG (A8592) was from Sigma-Aldrich, Ab for tubulin (F0601) was from SunGene, Abs for MITA (136474S), phospho-IRF3 (4947S), and phospho-IκBα (4947S) were obtained from Cell Singling Technology, anti-IRF3(sc-9082) and anti-IκBα (sc-371) were purchased from Santa Cruz Biotechnology. Staining Abs against CD3, CD4, CD8, CD19, CD25, CD44, CD62L, CD11c, CD11b, and F4/80 were purchased from BioLegend or SunGene. Flow cytometry analysis was performed with the instrument of BD FACSCelesta.

Total RNA was extracted from cells using TRIzol (Invitrogen), and the first-strand cDNA was reversed transcribed with All-in-One cDNA Synthesis SuperMix (BioTool). Gene expression was examined with a Bio-Rad CFX Connect system by a fast two-step amplification program with 2× SYBR Green Fast qPCR Master Mix (BioTool). The value obtained for each gene was normalized to that of the gene encoding β-actin. Gene-specific primers have been described previously (26, 35). The ELISA kits for IFN-β, TNF, and IL-6 (BioLegend) and CCL5 (4ABio) were used to detect the indicated cytokines in the sera or in the supernatants of cultured cells.

The experiments were performed as previously described (26, 35). In brief, cells were lysed in Nonidet P-40 lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 1% protease and phosphatase inhibitor mixture (BioTool). Cell lysates were subjected to SDS-PAGE, and immunoblot analysis was performed with the appropriate Abs. For immunoprecipitation assays, the lysates were immunoprecipitated with the appropriate Abs, and the precipitants were washed three times with lysis buffer containing 500 mMNaCl, followed by immunoblot analysis. The Abs were diluted in 3–5% (wt/vol) fat-free milk (BD Biosciences) or 1% BSA (Sigma-Aldrich) in TBS (1:500–1:2000).

These experiments were performed as previously described (26, 35). For deubiquitination assays in cells, cells were lysed with the lysis buffer containing 10 mM N-Ethylmaleimide (100 μl), and the supernatants were denatured at 95°C for 5 min in the presence of 1% SDS by lysates. The denatured lysates were diluted with lysis buffer until the concentration of SDS reduced below 0.1%, followed by immunoprecipitation (denature-immunoprecipitation) with the indicated Abs. The immunoprecipitants were subject to immunoblot analysis with anti-K48–linked ubiquitin chains.

Mouse lung fibroblasts (MLFs) were isolated from mice that were 8–10 wk old. Lungs were minced and digested in calcium- and magnesium-free HBSS buffer supplemented with 10 mg/ml type I collagenase (Worthington) and 20 μg/ml DNase I (Sigma-Aldrich) for 3 h at 37°C with shaking. Cell suspensions were filtered through sterile mesh, and the filtered cells were cultured in DMEM containing 10% FBS, 1% streptomycin and penicillin, and 10 μMβ-mercaptoethanol. Two days later, adherent fibroblasts were rinsed with HBSS and cultured for experiments. Bone marrow cells were isolated from mouse femur. The cells were cultured in DMEM containing 10% FBS, 1% streptomycin and penicillin, and 10 μMβ-mercaptoethanol, with M-CSF (10 ng/ml; PeproTech) for bone marrow–derived macrophage (BMDM) differentiation or GM-CSF (20 ng/ml; PeproTech) for bone marrow–derived dendritic cell (BMDC) differentiation. The medium was changed every 3 d. On day 7, cells were used for subsequent analysis.

For quantitative RT-PCR (qRT-PCR) or immunoblot analysis, cells seeded into 24-well plates (2–5 × 10⁵ cells per well) or six-well plates (10⁶–10⁷ cells per well) were infected with various viruses for the indicated time points. The variant of HSV-1, H129-G4, was previously described (34). For viral replication assays, cells (2–5 × 10⁵) were infected with HSV-1 or H129-G4. One hour later, the supernatants were removed and cells were washed with prewarmed PBS (1 ml) twice, followed by culture in full medium for 12–24 h. Viral replication was analyzed by flow cytometry, fluorescent microscopy, or qRT-PCR analysis. For mice infection, age- and sex-matched Usp20^+/+ and Usp20^−/− littermates were injected with HSV-1 (2 × 10⁶ PFU per mouse) and the survival of animals was monitored for 7 d. The lungs and brains were collected for qRT-PCR analysis or plaque assays at 24 h or 4 d postinfection, respectively.

For plaque assay, briefly, viral samples were serially diluted and incubated with Vero in the plate for 1 h, the homogenates or the dilutions were removed, and the infected Vero cells were washed with prewarmed PBS twice, followed by incubation with DMEM containing 1.5% methylcellulose for 48 h. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min before counting the plaques.

HEK293 cells were transfected with phage-6tag-MITA, phage-6tag-USP20, phage-6tag-USP20(C560/563S), phage-6tag-Cherry-MITA, phage-6tag-GFP-USP20, or the empty vector along with the packaging vectors pSPAX2 and pMD2G. The medium was changed with fresh full medium (10% FBS, 1% streptomycin/penicillin, and 10 μM 2-ME) after 8 h. Forty hours later, the supernatants were harvested to infect cells followed by various analyses.

Differences between experimental and control groups were tested using Student t test or two-way ANOVA with Bonferroni posttest. The p values <0.05 were considered statistically significant. For animal survival analysis, the Kaplan–Meier method was used to generate graphs, and the survival curves were analyzed with log-rank analysis.

We have previously investigated the deubiquitinating regulation of MITA and demonstrated that USP20 deubiquitinates and stabilizes MITA after HSV-1 infection (26). In this study, we initially mapped the domains of USP20 interacting with MITA and found that the UCH domain (aa 150–700) was associated with MITA (Fig. 1A). To further investigate the role of USP20 in antiviral signaling in vivo, we generated USP20-deficient mice by CRISPR/Cas9–mediated genome editing (Supplemental Fig. 1A). The generated mice lacked the fourth to the ninth exons of Usp20, which resulted in the early translational termination of USP20 (aa 1–170) (Supplemental Fig. 1B). The Usp20^−/− mice were normal in growth and development and were born in accordance with Mendelian ratio (Supplemental Fig. 1C), indicating that USP20 is dispensable for growth or development of mice. Flow cytometry analysis suggested that knockout of USP20 in mice did not affect the lymphocytes numbers or percentages in thymus, spleen, or peripheral lymph nodes (Supplemental Fig. 1D–F). In addition, knockout of USP20 did not affect the differentiation of BMDMs or BMDCs in M-CSF or GM-CSF cultures, respectively (Supplemental Fig. 1G), suggesting that USP20 is not required for the development and homeostasis of lymphocytes in vivo. We next examined the cellular localization of USP20 and MITA by transfecting GFP-tagged USP20 and Cherry-tagged MITA into Usp20^−/− BMDCs. The results suggest that a portion of USP20 and MITA was colocalized in the cytosol after HSV-1 infection (Fig. 1B). HSV-1 infection leads to puncta formation of MITA, a hallmark of MITA activation (36). Interestingly, we found that knockout of USP20 did not affect the puncta formation of MITA after HSV-1 infection (Fig. 1C).

We next examined whether USP20 plays a role in DNA virus-triggered expression of downstream genes with Usp20^+/+ and Usp20^−/− cells. Results from qRT-PCR analysis showed that the induction of Ifnb, Ip10, Il6, or Isg56 was significantly decreased in Usp20^−/− BMDCs and BMDMs compared with the wild-type counterparts after infection with HSV-1 or transfection of various DNA ligands (Fig. 2A, 2B). Immunoblot analysis suggested that HSV-1–induced phosphorylation of IRF3, IκBα, USP18, and p65 was substantially impaired in Usp20^−/− BMDCs or BMDMs compared with the wild-type counterparts (Fig. 2A, Supplemental Fig. 2). In addition, the production of IFN-β and TNF was also significantly compromised by knockout of USP20 in BMDCs and BMDMs (Fig. 2C). Moreover, the replication of HSV-1 was potentiated in Usp20^−/− BMDCs or BMDMs compared with the wild-type counterparts as monitored by the expression of HSV-1 UL30 gene, the HSV-1 titers in the supernatants, or the GFP percentages of H129-G4 viruses (37) (Fig. 2D–F). These data together suggest that USP20 positively regulates DNA virus-triggered signaling in various primary mouse cells.

We next examined whether USP20 plays a role in RNA virus-triggered signaling. The qRT-PCR analysis showed that Sendai virus (SeV)- or encephalomyocarditis virus (EMCV)-induced expression of Ifnb, Ip10, and Il6 was comparable in USP20-deficient BMDCs and MLFs compared with the wild-type cells (Fig. 3A, 3B). Consistently, knockout of USP20 did not inhibit SeV- or EMCV-induced phosphorylation of IRF3 or IκBα (Fig. 3A, 3B). In addition, intracellular poly(I:C)–induced expression of Ifnb, Ip10, and Il6 was not affected by knockout of USP20 (Fig. 3C). These data together suggest that USP20 is not essential for RNA virus-triggered signaling.

Next, we determined whether USP20 affects type I IFN–mediated antiviral responses. Wild-type and Usp20^−/− MLFs were infected with H129-G4 or HSV-1 for 1 h, and the cells were washed with prewarmed PBS for twice and cultured in fresh complete medium in the presence or absence of IFN-α (400 ng/ml) for 24 h, followed by fluorescent microscopy, flow cytometry, or qRT-PCR analyses. The results showed that the GFP intensities or percentages or the mRNA levels of HSV-1 UL30 gene were comparable between the wild-type and Usp20^−/− cells (Supplemental Fig. 3A, 3B). In addition, knockout of USP20 had no effect on IFN-α–induced phosphorylation of STAT1 (Supplemental Fig. 3C). These data together suggest that USP20 does not regulate type I IFN signaling or type I IFN–mediated antiviral responses.

We next examined the function of USP20 in DNA virus infection in vivo. Age- and gender-matched Usp20^+/+ and Usp20^−/− mice were i.v. injected with HSV-1 and monitored daily for eight successive days. Consistent with the results from gene induction analysis, Usp20^−/− mice were more susceptible to lethal HSV-1 infection than the control littermates (Fig. 4A). In addition, the induction of IFN-β, IL-6, and CCL5 was significantly decreased in the sera of Usp20^−/− mice compared with Usp20^+/+ mice at 12 h after HSV-1 infection (Fig. 4B). The expression of Ifnb and proinflammatory cytokines was severely impaired and the replication of HSV-1 (as monitored by the expression of UL30 gene) was exacerbated in lungs and brains from Usp20^−/− mice compared with Usp20^+/+ mice at 24 h or 4 d after HSV-1 infection, respectively (Fig. 4C, 4D). Results from plaque assays further confirmed that USP20 deficiency led to increased HSV-1 titers in the lungs and brains from Usp20^−/− mice 24 h or 4 d postinfection, respectively, compared with wild-type controls (Fig. 4E). These results together suggest that USP20 positively regulates virus-induced expression of downstream genes and is essential for host defense against DNA viruses in vivo.

We have previously shown that the DUB activity of USP20 is required for deubiquitination of MITA (26). To confirm the essential roles of USP20 DUB activity in regulation of HSV-1–triggered signaling in vivo, we transfected the empty vector, USP20 or its enzymatic inactive mutant USP20(C560/563S) [designated as USP20(CS)] into Usp20^−/− cells followed by infection with HSV-1 infection or transfection of ISD. Results from qRT-PCR and ELISA analysis showed that HSV-1 or cytosolic DNA–induced expression of Ifnb and Ip10 and production of IFN-β and TNF were substantially rescued in Usp20^−/− MLFs reconstituted with USP20 but not in those reconstituted with USP20(CS) (Fig. 5A, 5B). In addition, HSV-1–induced phosphorylation of IRF3 or IκBα was increased by the reconstitution of USP20 but not USP20(CS) into Usp20^−/− MLFs (Fig. 5C). Consistently, replication of HSV-1 was potentiated in Usp20^−/− MLFs reconstituted with USP20 but not in those reconstituted with USP20(CS) as monitored by GFP signals or the HSV-1 titers in the supernatants (Fig. 5D, 5E). These data together suggest that USP20-mediated potentiation of DNA virus-triggered signaling requires its deubiquitinating enzymatic activity.

We have previously demonstrated that knockdown of USP20 leads to increased K48-linked ubiquitination of MITA in THP-1 cells after HSV-1 infection (26). Consistent with these observations, HSV-1–induced K48-linked ubiquitination of MITA were substantially increased in Usp20^−/− MLFs compared with Usp20^+/+ MLFs (Fig. 6A). Reconstitution of USP20 but not USP20(CS) into Usp20^−/− MLFs impaired HSV-1–induced K48-linked ubiquitination of MITA (Fig. 6B), indicating that USP20 removes K48-linked polyubiquitin chains from MITA after viral infection, which may control the protein stability of MITA. In support of this notion, knockout of USP20 promoted degradation of MITA in the absence or presence of CHX (100 μg/ml) after infection with HSV-1 (Fig. 6C). In addition, HSV-1– or cytoplasmic DNA–induced degradation of MITA was blocked by the proteasome inhibitor MG132 but not the autophagy inhibitor 3MA in Usp20^−/−MLFs (Fig. 6D). These data collectively suggest that USP20 rescues MITA from proteasome-dependent degradation of MITA by deconjugating K48-linked ubiquitination of MITA.

Because the degradation of MITA is accelerated in USP20-deficient cells, we hypothesized that supplementation of MITA into USP20 knockout cells would restore virus-triggered expression of downstream genes. As we expected, reconstitution of MITA into Usp20^−/− MLFs rescued the phosphorylation of IRF3 and IκBα and the expression of Ifnb, Il6, and Ip10 after HSV-1 infection (Fig. 6E, 6F). Consistently, the replication of HSV-1 was suppressed in Usp20^−/− MLFs reconstituted with MITA as monitored by the GFP signals of H129-G4 or the HSV-1 titers in the supernatants (Fig. 6G, 6H). Together, these data suggest that MITA is the major target of USP20 in regulating cellular antiviral responses to DNA virus infection.

The adaptor protein MITA critically mediates cellular antiviral responses and autoimmunity. The activity and availability of MITA are strictly regulated by various PTMs including ubiquitination (38). Whereas the E3 ubiquitin ligases RNF5 and TRIM29 have been reported to catalyze K48-linked ubiquitination and proteasomal degradation of MITA (20–22), the DUB enzymes antagonizing such a modification remain to be identified. We have previously reported that deficiency of USP18 results in destabilization of MITA in a manner independent of its enzyme activity (26). Interestingly, USP20 is a direct DUB enzyme targeting MITA for deubiquitination in vitro and in THP-1 cells (26). In this study, we generated USP20-deficient mice and provided genetic evidence that deletion of USP20 in primary mouse cells promoted HSV-1–induced K48-linked ubiquitination and proteasomal degradation of MITA. Consistent with these observations, we found that knockout of USP20 impaired phosphorylation of IκBα and IRF3 and expression of downstream type I IFNs and proinflammatory cytokines after HSV-1 infection or cytoplasmic DNA challenge, and the USP20-deficient mice were more susceptible to lethal HSV-1 infection. In addition, we further demonstrated that complementation of MITA into Usp20^−/− cells fully restored HSV-1–induced phosphorylation of IκBα and IRF3 and induction of type I IFNs and proinflammatory cytokines. These data together suggest that USP20 targets MITA for deubiquitination and thereby stabilizes MITA to promote cellular antiviral responses.

The protein stability of MITA is controlled at multiple levels. Upon activation, MITA is rapidly transported to ERGIC, where it recruits TBK1 and IRF3 and promotes phosphorylation of IRF3 by TBK1 (39). This process requires iRhom2, which facilitates the trafficking and simultaneously recruits EIF3S5 to prevent the degradative ubiquitination of MITA (36). There are also reports showing that MITA traffics to autophagosomes that associate with endosomal compartments containing NF-κB and IRF3, where MITA recruits TBK1 and IKKα/β/γ to activate IRF3 and NF-κB (11). After its activation of IRF3, ULK1 phosphorylates and promotes the degradation of MITA, presumably through the autophagy pathway (12). In addition, TRIM38 and SENP2 catalyze sumoylation and desumoylation of MITA at the early and late phase of HSV-1 infection, thereby regulating the degradation of MITA through chaperon-mediated autophagy pathway (14). More recently, two groups have reported that TRIM29 and MITA form puncta in the cytosol, where TRIM29 induces K48-linked ubiquitination and proteasome-dependent degradation of MITA after HSV-1 infection or cytoplasmic DNA challenge (21, 22). Interestingly, we have previously shown that RNF5 ubiquitinates and induces degradation of MITA in the membrane fractions after viral infection (20). Our data suggest that USP20 removes K48-linked ubiquitin chains from MITA and keeps MITA away from proteasome-mediated degradation. This process is likely following the ubiquitination and puncta formation of MITA and it is highly possible that USP20 functions as a corrector that antagonizes excessive degradation of MITA mediated by E3 ubiquitin ligases after viral infection.

Currently, it is unlikely that USP20 regulates HSV-1 entry into cells. First, when treated with IFN-α, H129-G4 (GFP-expressing HSV-1) or HSV-1 replicated equally in Usp20^+/+ or Usp20^−/− MLFs as monitored by the GFP percentages or HSV-1 UL30 gene expression. It should be noted that USP20 did not regulate type I IFN–induced phosphorylation of STAT1, suggesting that the equal replication is more possible because of equal infection efficiency than USP20-mediated regulation of type I IFN–triggered signaling. Second, reconstitution of MITA into Usp20^−/− MLFs inhibited HSV-1 or H129-G4 replication as equally as did Usp20^+/+ MLFs, indicating that MITA is the major and sole factor for increased HSV-1 replication in Usp20^−/− cells. Finally, our previous and current studies suggest that knockdown or knockout of USP20 results in increased K48-linked ubiquitination and accelerated degradation of MITA. It is widely acknowledged that the primary role of MITA is to inhibit viral replication rather than viral entry by inducing type I IFNs production. However, whether USP20 affects virus entry requires more direct evidence and further investigations.

In this study, we found that the protein stability of MITA is very stable in resting cells, and HSV-1 infection or cytoplasmic DNA challenge rapidly induces its degradation in Usp20^−/− but not Usp20^+/+ cells, indicating essential roles of USP20 in protecting MITA from degradation. Several studies have reported various gain-of-function mutants of MITA that are associated with autoimmune diseases termed as STING-associated vasculopathy with onset in infancy (SAVI) (39–43). These mutants are activated spontaneously without cGAMP binding and constitutively undergo dimerization or oligomerization for activation. However, it is unknown whether these mutants are constitutively ubiquitinated at cellular compartments. Considering that deletion of MITA rescues lethality of Trex1^−/− or Rnaseh2 loss-of-function mutation knock-in mice (44–46), it is very interesting to investigate whether USP20 plays a role in autoimmunity by catalyzing deubiquitination and promoting the degradation of MITA.

We thank members of Zhong laboratory and the core facilities of the Medical Research Institute for technical help.

The authors have no financial conflicts of interest.