USP27X Deubiquitinates and Stabilizes the DNA Sensor Cgas to Regulate Cytosolic DNA−Mediated Signaling

Cutting Edge: USP27X Deubiquitinates and Stabilizes the DNA Sensor cGAS to Regulate Cytosolic DNA−Mediated Signaling

This information is current as Yunyun Guo, Fei Jiang, Lingli Kong, Bingqing Li, Yinlong of October 1, 2021. Yang, Lei Zhang, Bingyu Liu, Yi Zheng and Chengjiang Gao J Immunol 2019; 203:2049-2054; Prepublished online 18 September 2019; doi: 10.4049/jimmunol.1900514 http://www.jimmunol.org/content/203/8/2049 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/09/17/jimmunol.190051

Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 20 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/203/8/2049.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: USP27X Deubiquitinates and Stabilizes the DNA Sensor cGAS to Regulate Cytosolic DNA–Mediated Signaling Yunyun Guo,* Fei Jiang,* Lingli Kong,* Bingqing Li,† Yinlong Yang,† Lei Zhang,* Bingyu Liu,* Yi Zheng,* and Chengjiang Gao* Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA disease (5, 6). Thus, the cGAS pathway must be tightly regulated sensor, catalyzes the formation of the second messenger to prevent harmful activity arising from unrestrained signaling. 2939-cGAMP that binds to STING and triggers the Various posttranslational modifications modulate cGAS activity. type I IFN signaling. Activation of cGAS can be modu- The interaction between endoplasmic reticulum ubiquitin ligase lated by several protein posttranslational modifications, RNF185 and cGAS specifically catalyzes the K27-linked poly- including ubiquitination. However, the cGAS activation ubiquitination of cGAS, which promotes its enzymatic activity Downloaded from regulated by protein deubiquitination remains poorly un- (7). TRIM56 induces the K335 monoubiquitination of cGAS, derstood. In this study, we identified that deubiquitinase resulting in a marked increase of its dimerization, DNA-binding USP27X could interact with cGAS and cleave K48-linked activity, and cGAMP production (8). TRIM38 catalyzes the polyubiquitination chains from cGAS, leading to cGAS sumoylation at K217 of cGAS in uninfected cells and dur- stabilization. Consistently, knockout of Usp27x in mice ing the early phase of viral infection, which prevented the polyubiquitination-dependent degradation of cGAS to facilitate http://www.jimmunol.org/ macrophages resulted in an accelerated turnover of cGAS, an effective innate immune response to DNA viruses (9). Deu- decreased cGAMP production, phosphorylation of TBK1 b biquitination and ubiquitination are equally important in the and IRF3, and IFN- production. Furthermore, Usp27x normal functioning of organisms. Approximately 95 deubiqui- knockout mice macrophages showed impaired innate an- tinating enzymes (DUBs) are encoded in the human genome tiviral responses against HSV type 1 infection. Our data that falls into five families, but only 79 of them are predicted to suggest that USP27X is a novel regulator of the cGAS– be active deubiquitinases (10). In this regard, TRIM14 is found STING cytosolic DNA sensing pathway. The Journal to recruit USP14 to cleave K48-linked ubiquitination of cGAS at

of Immunology, 2019, 203: 2049–2054. K414 and mediate type I IFN signaling as well as antiviral im- by guest on October 1, 2021 munity through the cross-talk with autophagy (11). However, it is not clear whether other DUBs can regulate cGAS deubiqui- ctivation of the innate immune system relies on several tination directly and further participate in innate immunity. In classes of germline-encoded pattern-recognition re- this study, we demonstrated that USP27X directly interacts with ceptors, including TLRs, NOD-like receptors, retinoic A cGAS and uses its enzymatic activity to cleave K48-linked pol- acid–inducible gene I–like receptors (RLRs), and DNA sensors yubiquitin chains of cGAS, leading to the stabilization of cGAS (1, 2). Cyclic GMP-AMP (cGAMP) synthase (cGAS), which protein. Importantly, we found that macrophages bearing the belongs to the nucleotidyltransferase family, is one of the most deletion of the Usp27x gene had less production of IFN-b and important DNA sensors (3). Upon binding to DNA molecules, hence were more susceptible to HSV type 1 (HSV-1) infection. cGAS uses GTP and ATP to produce 2939-cGAMP, then Thus, our findings demonstrate that USP27X contributes to cGAMP binds to the endoplasmic reticulum protein STING innate immunity by directly regulating cGAS deubiquitination. (also known as MITA, ERIS), leading to IRF3 activation and IFN-b production (4). Materials and Methods The cGAS–STING pathway is essential for the host to Reagents and Abs protect the host against a large variety of DNA-containing cGAMP and polyinosinic:polycytidylic acid (poly[I:C]) were purchased from pathogens. However, aberrant activation of the cGAS pathway Invivogen. cGAMP and poly(I:C) were used at a final concentration of 50 and by self-DNA can also lead to autoimmune and inflammatory 10 mg/ml, respectively. HSV-60 is a 60-bp oligonucleotide containing viral

*Key Laboratory of Infection and Immunity of Shandong Province and Depart- Address correspondence and reprint requests to Dr. Chengjiang Gao, Department of ment of Immunology, School of Biomedical Sciences, Shandong University, Immunology, School of Biomedical Sciences, Shandong University, No. 44 Wenhua Xi Jinan, Shandong 250012, People’s Republic of China; and †Key Laboratory of Road, Jinan, Shandong 250012, China. E-mail address: [email protected] Rare and Uncommon Diseases, Department of Microbiology, Institute of Basic The online version of this article contains supplemental material. Medicine, Shandong First Medical Sciences, Jinan, Shandong 250062, People’s Republic of China Abbreviations used in this article: cGAMP, cyclic GMP-AMP; cGAS, cyclic GMP-AMP synthase; DUB, deubiquitinating enzyme; HA, hemagglutinin; HSV-1, HSV type 1; IP, ORCIDs: 0000-0002-4322-3744 (B. Li); 0000-0002-7228-5450 (B. Liu); 0000-0002- immunoprecipitation; ISD, IFN stimulatoryDNA;MOI,multiplicityofinfection; 9365-4497 (C.G.). poly(I:C), polyinosinic:polycytidylic acid; RLR, retinoic acid–inducible gene I–like receptor; Received for publication May 9, 2019. Accepted for publication August 16, 2019. SeV, Sendai virus; siRNA, small interfering RNA; VSV, vesicular stomatitis virus.

This work was supported by grants from the National Natural Science Foundation of Ó China (3173002 and 81525012 to C.G.). Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900514 2050 CUTTING EDGE: USP27X DEUBIQUITINATES cGAS

DNA motifs derived from the HSV-1 genome. Rabbit anti–c-Myc Ab and 1.5 ng/ml perfringolysin O for 1.5 h at 30˚C. Cells were lysed by (A190-105A) was from Bethl Laboratories; rabbit anti-Flag Ab (PA1-984B) adding 0.2% Nonidet P-40 and subjected to native gel electrophoresis. and rabbit anti-USP27X Ab (PA5-70389) were from Thermo Fisher Scien- IRF3 dimerization was detected. tific; rabbit anti-hemagglutinin (HA) Ab was from Rockland; rabbit anti-cGAS Ab (D3080), mouse anti-Ub (P4D1), rabbit anti-P65 (D14E12), anti-pP65 Plaque assays and detection of virus replication k k b b 6 (93H1), anti-I B (44D4), anti-pI B (14D4), anti-IKK (D30C6), anti-pIKK The WT or Usp27x-KO RAW264.7 cells (2 3 10 ) were infected with VSV (C84E11), anti-IRF3 (D83B9), anti-pIRF3 (4D46), anti-TBK1 (3031S), (multiplicity of infection [MOI] = 0.1) or HSV-1 (MOI = 10) for the in- and anti-pTBK1 (D52C2) were from Cell Signaling Technology; mouse anti- dicated times. HSV and VSV plaque assay and replication were performed FLAG (M2) Abs were from Sigma-Aldrich; and HRP-conjugated goat anti- as described previously (12). mouse or rabbit IgG Abs used for Western blotting were from Calbiochem. Statistical analysis Preparation of mouse peritoneal macrophages All data are presented as mean 6 SD of one representative experiment. Peritoneal macrophages were harvested and cultured as previously described Statistical significance was determined with the one-way ANOVA, with a (12). p value ,0.05 considered statistically significant. Cells and viruses Results and Discussion HEK293T and RAW264.7 macrophages were obtained from the American USP27X regulates cGAS deubiquitination Type Culture Collection. Sendai virus (SeV) was purchased from the China Center for Type Culture Collection (Wuhan University, China). Vesicular Previous studies have demonstrated that cGAS is modified by stomatitis virus (VSV) and HSV-1 were provided by H. Meng (Institute of various types of ubiquitin chains, which is essential for antiviral Basic Medicine, Shandong Academy of Medical Sciences, China). immunity (7–9), whereas the cGAS deubiquitination is not

Generation of Usp27x-KO RAW264.7 macrophage well deﬁned. To identify the DUBs that potentially regulate Downloaded from through CRISPR-Cas9 cGAS deubiquitination, we transfected DUB expression plasmids To obtain the Usp27x-KO RAW264.7 cell lines, CRISPR-Cas9 genomic into HEK293T cells together with Flag-cGAS and HA-ubiquitin editing for gene deletion was used, as previously described (13). Guide RNA expression plasmids, then the ubiquitination of cGAS was were designed and cloned into the vector lentiCRISPRv2. The guide RNA analyzed through IP and Western blotting. We found that 9 sequence used is as follows: Usp27x sg 5 -CACCGAATTAGTCCTCG- USP27X overexpression potentially attenuated cGAS ubiq- TAAGCCGA-39.

uitination(Supplemental Fig. 1). http://www.jimmunol.org/ Plasmid constructs and small interfering RNA To investigate whether attenuated cGAS ubiquitination The coding region of human USP27X and cGAS was amplified from human depends on the enzymatic activity of USP27X, we transfected cDNA and subcloned into pCDNA3.1. The coding region of mouse Usp27x WT USP27X or its enzymatic activity mutant C87A into was amplified from mouse cDNA and subcloned into pLVX-IRES-Puro. HEK293T cells together with Flag-tagged cGAS and HA- HA-ubiquitin and other plasmids were described previously (12). The corresponding empty vectors were used as negative controls in all trans- ubiquitin, and the cGAS ubiquitination was examined. The fection experiments. The small interfering sequences against mice were as WT USP27X but not the mutant C87A led to a significant follows: Usp27x:59-GCGCTAGAGTCCTGCATA-39,59-GCTCACTAT- reduction of cGAS ubiquitination, indicating that attenuated 9 GAAGAAGTTA-3 . Scramble small interfering RNA (siRNA) were used as a cGAS ubiquitination requires USP27X enzymatic activity negative control in all RNA interference experiments. by guest on October 1, 2021 (Fig.1A).Wealsoperformedaninvitrodeubiquitination ELISA assay with recombinant USP27X protein. In this experi- The concentrations of IFN-b in culture supernatants were measured by ELISA ment, ubiquitinated cGAS was first immunoprecipitated kits (R&D Systems). The concentration of 29-39-cGAMP was measured by from HEK293T cells transfectedwithFlag-cGASandHA- 9 9 2 -3 -cGAMP ELISA kit (Cayman Chemical). ubiquitin plasmids, then recombinant USP27X was added Lentivirus preparation in the in vitro deubiquitination assays. As shown in Fig. 1B, Usp27x sequence was subcloned into pLVX-IRES-Puro vector, and the empty recombinant WT USP27X protein but not the mutant C87A pLVX-IRES-Puro was used as a control. The lentivirus was produced by protein was able to decrease cGAS ubiquitination. To exclude transient transfection of the pLVX-IRES-Puro-Usp27x construct or control any artificial effect caused by overexpression and determine vector into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) whether USP27X-mediated cGAS deubiquitination occurs in with pLVX-IRES-Puro, pMD2.G, and psPAX2. physiological conditions, we used CRISPR/Cas9 technology In vitro deubiquitination assay with a guide RNA specific to the mouse Usp27x gene to es- Recombinant Myc-USP27X was expressed with a TNT Quick Coupled tablish Usp27x-KO RAW264.7 macrophages and examined Transcription/Translation System (Promega), according to the instructions the endogenous cGAS ubiquitination. DNA sequencing and of the manufacturer. The in vitro deubiquitination assay was performed as Western blot confirmed the success of Usp27x knockout in previously described (14). RAW264.7 macrophages (Supplemental Fig. 2A, 2B). The level In vitro binding assay of endogenous cGAS ubiquitination was obviously increased in Myc-USP27X and Flag-cGAS proteins were expressed with a TNT Quick Usp27x-KO RAW264.7 cells compared with that in WT cells Coupled Transcription/Translation System (Promega), according to the in- upon IFN stimulatory DNA (ISD) stimulation (Fig. 1C). We structions of the manufacturer. Myc-USP27X and Flag-cGAS were mixed, also demonstrated that cGAS ubiquitination induced by HSV-1 followed by immunoprecipitation (IP) with Flag Abs and Western blotting with Myc Abs. infection was increased in Usp27x siRNA-transfected primary macrophages compared with that in control siRNA-transfected In vitro assay for cGAMP activity macrophages (Supplemental Fig. 1I). All together, these data The cGAMP activity assay was performed as described previously (15). suggest that USP27X regulates cGAS deubiquitination in a RAW264.7 cells were transfected with HSV-60 for indicated time points manner that is dependent on its DUB activity. and then homogenized by douncing in the hypotonic buffer. The homogenate was centrifuged at 100,000 rpm for 5 min. Then the supernatant was heated USP27X interacts with cGAS at 95˚C for 5 min and centrifuged again at 12,000 rpm for 5 min to remove denatured proteins. The heat-resistant supernatant was incubated with 1 3 106 To further investigate how USP27X regulates cGAS deubi- RAW264.7 cells in an 8-ml reaction containing 2 mM ATP, 1 U/ml benzonase, quitination, the interaction between these two proteins was The Journal of Immunology 2051

and HA-ubiquitin or its lysine residue mutants with or without USP27X into HEK293T cells. As shown in Fig. 2A, overexpression of USP27X significantly decreased cGAS ubiquitination in WT and K48 ubiquitin transfected cells, indicating that USP27X mainly cleaved K48-linked polyubiquitin chain from cGAS. K48-linked ubiquitination usually leads to the degradation of target proteins through proteasome. Thus, we studied whether USP27X could inhibit cGAS protein degradation. Consistently, overexpression of WT USP27X in HEK293T cells increased Flag-cGAS protein level in a dose-dependent manner (Fig. 2B). In contrast, the cGAS protein level was not changed in C87A transfected cells (Fig. 2B). To further confirm USP27X regulates cGAS stability, we infected WT and Usp27x-KO RAW264.7 macrophages with HSV-1 and measured the cGAS protein expression. As shown in Fig. 2C, cGAS protein level was substantially decreased in Usp27x-KO macrophages compared with WT counterparts upon HSV-1 infection. Similarly, cGAS protein level was decreased in primary peritoneal macrophages transfected with Usp27x siRNA Downloaded from compared with those transfected with control siRNA (Fig. 2D). To directly confirm that USP27X regulates cGAS degradation, FIGURE 1. USP27X interacts with cGAS and regulates its deubiquitination. WT and Usp27x-KO RAW264.7 macrophages were first in- (A) Flag-cGAS and HA-ubiquitin (HA-Ub) were transfected into HEK293T cells together with USP27X or C87A, and cell lysates were subjected to IP with fected with HSV-1 for 4 h, then the protein synthesis inhibitor anti-Flag Ab, followed by Western blotting with HA Ab. (B) HEK293T cells cycloheximide was used to treat the cells for different times. http://www.jimmunol.org/ were transiently transfected with Flag-cGAS and HA-Ub, then ubiquitinated As shown in Fig. 2E, deficiency of Usp27x greatly accelerated cGAS was enriched with anti-Flag Ab. In vitro synthesized USP27X was in- the degradation of cGAS in RAW264.7 cells. Interestingly, cubated together with ubiquitinated cGAS in deubiquitylation assay buffer at we found that decreased cGAS protein levels in Usp27x-KO 37˚C for 1 h. The ubiquitination of cGAS was analyzed by Western blotting. C RAW264.7 cells could be partially restored by treatment of ( ) Usp27x knockout and counterpart macrophages were transfected with ISD MG132 or chloroquine, indicating both proteasomal and and harvested at indicated times. The ubiquitinated cGAS was enriched with anti-cGAS Ab and detected with ubiquitin (Ub) Ab. (D) Co-IP was performed autophagic degradation pathways are involved in USP27X- in lysates of HEK293T cells expressing Flag-cGAS and Myc-USP27X mediated regulation of cGAS protein (Fig. 2F). All together, or C87A. (E) Flag-cGAS and Myc-USP27X were obtained by in vitro these data demonstrated that USP27X interacts with cGAS transcription and translation. Interaction between cGAS and Usp27x was assayed to remove K48-linked ubiquitination and enhance cGAS by guest on October 1, 2021 followed by IP with Flag Abs and Western blot with Myc Abs. (F) Co-IP was stability. performed in lysates of peritoneal macrophages transfected with HSV-60 for indicated times. Similar results were obtained in three independent USP27X positively regulates intracellular DNA-induced experiments. innate signaling measured. Co-IP with Flag Abs and Western blot with Recognition of cytosolic DNA by cGAS led to the production Myc Abs demonstrated that cGAS could interact with both of cGAMP, which binds to STING to promote the phos- USP27X WT and C87A in HEK293 cells (Fig. 1D). We also phorylation of TBK1 and IRF3, and then phosphorylated performed the in vitro binding assay with recombinant USP27X IRF3 dimerizes and translocates into the nucleus to induce type and cGAS proteins. Co-IP and Western blot demonstrated that I IFN production (18–20). HSV-1 infection and ISD trans- USP27X associates with cGAS directly (Fig. 1E). Further, we fection could induce phosphorylation of IRF3 and TBK1 showed that endogenous cGAS and USP27X could form a (Fig. 3A, Supplemental Fig. 2C). Phosphorylation of IRF3 complex; notably, this interaction was enhanced upon HSV-60 and TBK1 induced by HSV-1 infection and ISD transfection transfection (Fig. 1F). USP14 has been demonstrated to regulate was greatly decreased in Usp27x-KO RAW264.7 cells (Fig. 3A, cGAS deubiquitination (11). However, USP14 and cGAS Supplemental Fig. 2C). In contrast, cGAMP-induced phosphor- did not interact with each other directly; rather, USP14 ylation of IRF3 and TBK1 was barely affected in Usp27x-KO was recruited to cGAS through TRIM14. We found USP14 RAW264.7 cells (Fig. 3A). Similarly, TBK1 and IRF3 phos- could not decrease the cGAS ubiquitination in our screen- phorylation was also decreased in primary peritoneal macro- ing experiments (Supplemental Fig. 1). This discrepancy may phages transfected with Usp27x siRNA compared with those be caused by a lack of TRIM14 expression in HEK293T cells. transfected with control siRNA (Supplemental Fig. 3D). In- Taken together, our data suggest that USP27X directly interacts terestingly, SeV- and poly(I:C)-induced phosphorylation of with cGAS to regulate cGAS deubiquitination. IRF3 and TBK1 were increased at late time points, indicating USP27X may negatively regulate RLR- and TLR3-mediated USP27X regulates cGAS stabilization signaling (Supplemental Fig. 2D). HSV-1–induced phos- Protein ubiquitination can be divided into several forms phorylation of p65, IkB, and IKKb was also decreased in according to the lysine residue used to form polyubiquitin Usp27x-KO RAW264.7 cells (SupplementalFig.2E).To chains, and different forms of ubiquitination have different directly confirm that Usp27x deficiency affects the phosphor- functions (16, 17). To study the form of ubiquitin chains in ylation of IRF3 and TBK1 on HSV-1 infection, we restored cGAS that was cleaved by USP27X, we transfected Flag-cGAS Usp27x expression in Usp27x-KO macrophage through 2052 CUTTING EDGE: USP27X DEUBIQUITINATES cGAS

signaling by USP27X, SeV- and poly(I:C)-induced expression of Ifnb1 mRNA was increased in Usp27x siRNA-transfected macrophages (Supplemental Fig. 3E). Knockout of Usp27x in RAW264.7 macrophages resulted in decreased expression of Ifnb1 mRNA expression on ISD transfection and HSV-1 infection. In contrast, cGAMP transfection-induced Ifnb1 mRNA expression was comparable in Usp27x-KO and WT cells (Fig. 3D, Supplemental Fig. 2G). SeV- and poly(I:C)-induced expression of Ifnb1 mRNA was increased (Supplemental Fig. 2G). We further found that secretion of IFN-b in response to HSV-1 infection, but not to cGAMP stimulation, was sig- niﬁcantly impaired in Usp27x-KO RAW264.7 cells (Fig. 3E). Overexpression of WT Usp27x, but not the enzymatic mutant C87A in Usp27x-KO RAW264.7 cells, rescued HSV-1 infection-induced expression of Ifnb1 mRNA and IFN-b secretion (Fig. 3F). Collectively, these data indicated that USP27X is essential for the cytosolic DNA-induced innate signaling and IFN-b production. Downloaded from USP27X regulates cGAMP production cGAS is the enzyme responsible for the production of the second messenger cGAMP (3). To explore the function of USP27X-mediated deubiquitination and stability of cGAS, we measured cGAMP production in Usp27x-KO RAW264.7

cells after transfection with HSV-60. cGAMP levels in the http://www.jimmunol.org/ stimulated cells were indirectly measured through IRF3 phosphorylation and dimerization. As expected, culture super- FIGURE 2. USP27X regulates the stability of cGAS. (A)Flag-cGAS, HA-ubiquitin (HA-Ub), or its lysine residue mutants were transfected in natant from WT macrophages stimulated with HSV-60 induced HEK293T cells with or without Myc-USP27X. Co-IP assays were performed. IRF3 phosphorylation and dimerization, whereas the cul- (B) Flag-cGAS was transfected in HEK293T cells together with gradient ture supernatant from Usp27x-KO RAW264.7 induced less amount of Myc-USP27X or C87A. cGAS protein level was detected by IRF3 phosphorylation and dimerization (Fig. 4A), indi- Western blot. (C)WTandUsp27x-KO RAW264.7 macrophages were in- cating Usp27x-KO RAW264.7 cells produced less cGAMP fected with HSV-1 for the indicated times, then cGAS protein level was

on HSV-60 transfection compared with WT macrophages. by guest on October 1, 2021 detected. (D) Primary peritoneal macrophages were transfected with Usp27x We also measured the cGAMP production in Usp27x-KO siRNA or scramble siRNA, infected with HSV-1 for the indicated times, 9 9 and then cGAS protein level was detected by Western blot. (E)WTand cells after HSV-60 transfection using 2 3 -cGAMP ELISA Usp27x-KO RAW264.7 macrophages were infected with HSV-1 for 4 h, Kit. Similar to the data of cGAMP-induced IRF3 activation, then treated with cycloheximide (100 mg/ml) for the indicated times. cGAMP production was decreased in Usp27x-KO cells com- cGAS protein level was detected by Western blot. (F) WT and Usp27x-KO pared with those in WT macrophages (Fig. 4B). Importantly, RAW264.7 cells were pretreated with MG132 or chloroquine (CQ) for 6 h, restoration of the WT Usp27x expression, but not the mutant followed by HSV-1 stimulation for the indicated times. cGAS protein level C87A expression, in Usp27x-KO cells rescued HSV-60–induced was detected by Western blot. Quantiﬁcation of cGAS protein levels relative to b-actin is shown in (B)–(F). Similar results were obtained in three independent cGAMP production. All together, these data suggested that experiments. USP27X regulates cGAMP production through deubiquitination of cGAS protein. transduction with Usp27x overexpression lentivirus. Over- USP27X potentiates innate antiviral responses to DNA virus expression of WT Usp27x, but not the enzymatic mutant Finally, we assessed if USP27X played a role in the regulation C87A, rescued HSV-1–induced phosphorylation of IRF3 of DNA virus infection. WT and Usp27x-KO RAW264.7 cells and TBK1 (Fig. 3B). were infected with HSV-1, and then the virus titer in the culture We further measured IRF3 dimerization on various stimula- medium and genomic DNA copy of HSV-1 in the infected cells tions. Consistent with IRF3 phosphorylation, IRF3 dimerization were measured by plaque assay and quantitative PCR, respec- was also decreased in Usp27x-KO RAW264.7 cells transfected tively. As a control, VSV was also used to infect the WT and with HSV-1 or ISD compared with those in their control Usp27x-KO cells. Plaque assay of RAW264.7 cells infected with counterparts (Fig. 3C, Supplemental Fig. 2F). cGAMP-induced HSV-1 showed that knockout of Usp27x greatly increased viral IRF3 dimerization was not changed between WT and Usp27x-KO replication compared with that in WT infected cells (Fig. 4C). macrophages (Fig. 3C). Consistently, the HSV-1 genomic DNA copy number was To investigate whether Usp27x regulates IFN-b production, also signiﬁcantly increased in the Usp27x-KO cells compared peritoneal macrophages were transfected with a scramble siRNA with WT cells (Fig. 4D). Importantly, restoration of WT or Usp27x siRNA (Supplemental Fig. 3A). We found that knock- Usp27x expression, but not the mutant C87A expression in down of Usp27x expression greatly decreased Ifnb1 mRNA and Usp27x-KO cells, repressed HSV-1 replication (Fig. 4C, 4D). IFN-b secretion in HSV-1–infected or ISD-transfected cells but Knockdown of Usp27x expression in primary peritoneal not in cGAMP-stimulated cells (Supplemental Fig. 3B, 3C). macrophages through Usp27x siRNA also increased HSV-1 Consistent with negative regulation of RLR- and TLR3-mediated replication (Fig. 4E, 4F). In contrast, VSV replication was The Journal of Immunology 2053 Downloaded from

FIGURE 3. USP27X positively regulates intracellular DNA-induced innate signaling. (A) Western blot of phosphorylated (p) and total IRF3 and TBK1 in WT and Usp27x-KO RAW264.7 cells infected with HSV-1 (MOI = 10) or stimulated with cGAMP (50 mg/ml) for the indicated time points. (B) Western blot of http://www.jimmunol.org/ phosphorylated (p) and total IRF3 and TBK1 in WT, Usp27x-KO, and Usp27x-KO RAW264.7 cells with overexpression of WT Usp27x or C87A mutant infected with HSV-1 (MOI = 10). (C) Native PAGE analysis of IRF3 dimerization in WT and Usp27x-KO RAW264.7 cells infected with HSV-1 (MOI = 10) or stimulated with cGAMP (50 mg/ml) for the indicated time points. (D) Quantitative PCR of Ifnb1 mRNA in WT and Usp27x-KO RAW264.7 cells infected with HSV-1 or stimulated with cGAMP for indicated time points. (E) ELISA for IFN-b production in culture supernatant from WT and Usp27x-KO RAW264.7 cells infected with HSV-1 or stimulated with cGAMP. (F) Quantitative PCR and ELISA analysis of Ifnb1 mRNA and IFN-b production in WT, Usp27x-KO, and Usp27x-KO RAW264.7 cells with overexpression of WT Usp27x or C87A mutant infected with HSV-1 (MOI = 10). Data are shown as mean 6 SD of triplicates from one representative experiment in (D)–(F). Similar results were obtained in three independent experiments. **p , 0.01.

decreased in Usp27x-KO cells compared with that in WT cells with cGAS and cleave K48-linked ubiquitination from cGAS, by guest on October 1, 2021 (SupplementalFig.4A,4B),indicatingUsp27x positively leading to the stabilization of cGAS. Further, we deleted the regulates DNA virus infection but negatively regulates RNA Usp27x gene in RAW264.7 macrophages with CRISPR/Cas9 virus infection. technology to assess the role of Usp27x in cytosolic DNA- Previous study reported that TRIM14 deﬁciency impairs mediated innate immunity. We found that Usp27x deﬁciency type I IFN signaling by both HSV-1 and VSV infection via increased the K48-linked ubiquitination and turnover of cGAS recruiting USP14 to deubiquitinate cGAS (11). In our study, and impaired DNA virus-induced cGAMP production, IRF3 we demonstrated that USP27X could directly form a complex phosphorylation, and IFN-b production.

FIGURE 4. USP27X positively regulates cGAMP production and anti-DNA virus infection. (A)WTorUsp27x-KO RAW264.7 cells were stimulated with HSV-60 for indicated times, and extracts from these cells were used to prepare heat-resistant supernatants, which were delivered to permeabilized fresh RAW264.7 cells to stimulate IRF3 dimerization. (B) ELISA analysis of cGAMP production in WT, Usp27x-KO, and Usp27x-KO RAW264.7 cells with overexpression of WT Usp27x or C87A mutant stimulated with ISD transfection. (C and D) Plaque assay of HSV-1 titers and copy number of HSV-1 genomic DNA were measured in WT, Usp27x-KO, and Usp27x-KO RAW264.7 cells with overexpression of WT Usp27x or C87A mutant. (E and F) Plaque assay of HSV-1 titers and copy number of HSV-1 genomic DNA were measured in primary peritoneal macrophages transfected with Usp27x siRNA or control siRNA. Data are shown as mean 6 SD of triplicates from one representative experiment in (C)–(F). Similar results were obtained in three independent experiments. **p , 0.01. 2054 CUTTING EDGE: USP27X DEUBIQUITINATES cGAS

We also found that USP27X plays different roles in the 6. Gao, D., T. Li, X. D. Li, X. Chen, Q. Z. Li, M. Wight-Carter, and Z. J. Chen. 2015. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune control of DNA virus and RNA virus infection. During HSV-1 diseases. Proc. Natl. Acad. Sci. USA 112: E5699–E5705. infection, we found that USP27X targets cGAS to prevent its 7. Wang, Q., L. Huang, Z. Hong, Z. Lv, Z. Mao, Y. Tang, X. Kong, S. Li, Y. Cui, H. Liu, et al. 2017. The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated ubiquitination-dependent degradation; therefore, Usp27x- innate immune response. PLoS Pathog. 13: e1006264. deficient RAW264.7 macrophages were more permissive to 8. Seo, G. J., C. Kim, W. J. Shin, E. H. Sklan, H. Eoh, and J. U. Jung. 2018. HSV-1 infection. During VSV infection, USP27X may target TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing. Nat. Commun. 9: 613. other signaling molecules to negatively regulate RNA virus 9. Hu, M. M., Q. Yang, X. Q. Xie, C. Y. Liao, H. Lin, T. T. Liu, L. Yin, and infection. Thus, Usp27x deficiency in RAW264.7 macro- H. B. Shu. 2016. Sumoylation promotes the stability of the DNA sensor cGAS and the adaptor STING to regulate the kinetics of response to DNA virus. Immunity phages repressed VSV replication. The specific mechanism 45: 555–569. for USP27X in the negative regulation of RLR signaling needs 10. Komander, D., M. J. Clague, and S. Urbe´. 2009. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10: 550–563. further investigation. 11. Chen, M., Q. Meng, Y. Qin, P. Liang, P. Tan, L. He, Y. Zhou, Y. Chen, J. Huang, Overall, this study provides strong evidence that USP27X R. F. Wang, and J. Cui. 2016. TRIM14 inhibits cGAS degradation mediated by selective autophagy receptor p62 to promote innate immune responses. Mol. Cell is responsible for the deubiquitination of cGAS and sheds light 64: 105–119. on the regulation of cGAS activation. Given the fact that 12. Song, G., B. Liu, Z. Li, H. Wu, P. Wang, K. Zhao, G. Jiang, L. Zhang, and C. Gao. cGAS–STING pathways play an important role in innate 2016. E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1. Nat. Immunol. 17: 1342–1351. antiviral immunity and autoimmune diseases, USP27X may 13.Ran,F.A.,P.D.Hsu,J.Wright,V.Agarwala,D.A.Scott,andF.Zhang. have therapeutic potential for the prevention and treatment of 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8: 2281–2308. autoimmune diseases with aberrant DNA signaling. 14. Guo, Y. Y., Y. Lu, Y. Zheng, X. R. Chen, J. L. Dong, R. R. Yuan, S. H. Huang, H. Yu, Y. Wang, Z. Y. Chen, and B. Su. 2017. Ubiquitin C-terminal hydrolase L1 Downloaded from (UCH-L1) promotes hippocampus-dependent memory via its deubiquitinating Disclosures effect on TrkB. J. Neurosci. 37: 5978–5995. The authors have no financial conflicts of interest. 15. Gao, D., J. Wu, Y. T. Wu, F. Du, C. Aroh, N. Yan, L. Sun, and Z. J. Chen. 2013. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 341: 903–906. References 16. Davis, M. E., and M. U. Gack. 2015. Ubiquitination in the antiviral immune re- 1. Roers, A., B. Hiller, and V. Hornung. 2016. Recognition of endogenous nucleic sponse. Virology 479-480: 52–65.

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Screening DUBs that regulating cGAS ubiquitination. (A-H) The expression plasmids for indicated DUBs were transfected into HEK293T cells together with Flag-cGAS and HA-Ub. 48h later, cell lysates were subjected to Co-IP and western blotting analysis. (I) Primary macrophages were transfected with Usp27X siRNA or control siRNA followed with HSV-1 infection for 4 h. The ubiquitinated cGAS was enriched with anti-cGAS antibody and detected with Ub antibody. Similar results were obtained in three independent experiments.

Supplementary Figure2

Usp27x-KO RAW264.7 macrophages showed impaired DNA-induced innate signaling. (A) Western blot of Usp27x expression in WT and four stable Usp27x-KO clones. Knockout efficiency of Clone 4 is the highest and chosen in this study. (B) The DNA sequencing results of the WT and Usp27x-KO RAW264.7 macrophages Clone

4 are shown. (C, D) Western blot of phosphorylated (p-) and total IRF3 in WT and Usp27x-KO RAW264.7 cells stimulated with ISD, infected with SeV or stimulated with poly(I:C) for indicated times. (E) Western blot of phosphorylated (p-) and total IKK-β, IκB and P65 in WT and Usp27x-KO RAW264.7 cells infected HSV-1(MOI=10) for indicated times. (F) Native PAGE of IRF3 dimerization in WT and Usp27x-KO RAW264.7 cells stimulated with ISD for indicated times. (G) qPCR of Ifnb1 mRNA in WT and Usp27x-KO RAW264.7 cells transfected with ISD, infected with SeV or stimulated with poly(I:C) for indicated times. Data are shown as mean±SD of triplicates from one representative experiment (**, p<0.01). Similar results were obtained in three independent experiments. Supplementary Figure3

USP27X regulates DNA-induced innate signaling in primary macrophages. (A) qPCR of Usp27x mRNA in peritoneal macrophages transfected with scramble siRNA (Si scramble) or Usp27x-specific siRNA (si Usp27x) for 24 h. (B, C) qPCR of Ifnb1 mRNA and ELISA of IFN-β protein in peritoneal macrophages transfected with scramble siRNA or Usp27x-specific siRNA 1 for 24 h followed by HSV-1 infection or ISD, cGAMP or stimulation indicated times. (D) Western blot of phosphorylated (p-) and total IRF3, TBK1 in lysates of peritoneal macrophages transfected with scramble siRNA or Usp27x-specific siRNA 1 for 24 h followed with HSV-1 infection or ISD, cGAMP stimulation for indicated times. (E) qPCR analysis of Ifnb1 mRNA in peritoneal macrophages transfected with scramble siRNA or Usp27x-specific siRNA 1 for 24 h followed by SeV infection or poly(I:C) stimulation for indicated times. Data are shown as mean±SD of triplicates from one representative experiment in A, B, C and E (**, p<0.01). Similar results were obtained in three independent experiments. Supplementary Figure 4

USP27X negatively regulates VSV infection. (A and B) plaque assay of VSV titers (A) and qPCR analysis of VSV mRNA (B) in WT and Usp27x-KO cells infected with VSV (MOI=0.1) for various times. Data are shown as mean±SD of triplicates from one representative experiment. (**, p<0.01; *, p<0.05). Similar results were obtained in three independent experiments.