GNAQ Negatively Regulates Antiviral Innate Immune Responses in a -Dependent Manner

This information is current as Ning Wang, Hongjun Huang, Qingqing Xiong, Naiyang of October 3, 2021. Chen, Nanxi Xi, Peilun Wu, Mingyao Liu, Min Qian, Qin Wang and Bing Du J Immunol published online 19 July 2019 http://www.jimmunol.org/content/early/2019/07/18/jimmun ol.1900427 Downloaded from

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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. Published July 19, 2019, doi:10.4049/jimmunol.1900427 The Journal of Immunology

GNAQ Negatively Regulates Antiviral Innate Immune Responses in a Calcineurin-Dependent Manner

Ning Wang,*,1 Hongjun Huang,*,1 Qingqing Xiong,* Naiyang Chen,* Nanxi Xi,* Peilun Wu,* Mingyao Liu,* Min Qian,* Qin Wang,† and Bing Du*

Although guanine nucleotide-binding ()–coupled receptors (GPCRs) constitute the largest cell surface mem- brane receptor family and transduce thousands of extracellular signals into the cytoplasm, only four kinds of G protein a subunits (Gas, Gai/o, Gaq/11, and Ga12/13) are coupled to regulate cAMP or phosphatidylinositol signals. Growing evidence suggests that viruses tend to hijack GPCRs and harness their activated intracellular signaling pathways. Thus, understanding the roles of G protein signaling will further uncover the GPCR signaling pathways that are exploited by viruses. In this study, we demonstrate that the expression of GNAQ (Gq a subunit) was downregulated during viral infection and that small interfering RNA–mediated GNAQ knockdown protected host cells from both vesicular stomatitis virus (VSV) and HSV type 1 infection. Downloaded from Meanwhile, VSV and HSV type 1 replication was reduced significantly in Gnaq-deficient macrophages. Accordingly, the VSV distribution in the liver, spleen, and lung was reduced in Gnaq-deficient mice during VSV infection, and Gnaq-deficient mice were much more resistant to VSV infection than wild-type mice. Mechanistically, GNAQ limits type I IFN production through the canonical PLC-b/Ca2+/CALNA signaling pathway, which has been demonstrated to dephosphorylate virus-activated TANK-binding kinase 1 (TBK1). Thus, our data demonstrate that GNAQ negatively regulates the antiviral innate immune responses in a calcineurin-dependent manner. These findings also provide insights into the function and cross-talk of the classic http://www.jimmunol.org/ GPCR signaling pathway with antiviral innate immune responses and suggest a potential therapeutic role for GNAQ in controlling viral diseases. The Journal of Immunology, 2019, 203: 000–000.

nnate immunity is the first line of host defense against in- , which could interfere with different viruses through distinct vading pathogens. Type I IFN (IFN-I) is rapidly activated by mechanisms (2). Although sufficient IFN-I production is essential I the innate immune system during viral infection and broadly for efficient host defense against viral infection and triggers ap- inhibits viral infection (1). As a pivotal mediator of host defense optosis of virus-infected cells, excessive production of IFN-I may against virus challenges, IFN-I establishes a cellular antiviral state lead to tissue damage or immune disorders (3, 4). Hence, IFN-I mainly through the upregulation of hundreds of IFN-stimulated production during viral infection should be spatially and tempo- by guest on October 3, 2021 rally controlled to initiate an appropriate immune response that can eliminate invading viruses without immune damage. *Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, The cell surface guanine nucleotide–binding protein (G protein)– China; and †Department of Nephrology and Rheumatology, Shanghai Fengxian coupled receptors (GPCRs) are encoded by .1000 genes in the Central Hospital, Shanghai 201400, China and constitute the largest family of sensory recep- 1 These authors contributed equally to this work. tors; GPCRs are activated by a diverse array of ligands, including ORCID: 0000-0002-5402-6527 (B.D.). hormones, peptides, amino acids, ions and photons of light, and Received for publication April 12, 2019. Accepted for publication June 20, 2019. transduce signals through a wide range of effectors (5, 6). Inter- This work was supported by the National Key R&D Program of China (2018YFA0507001 estingly, growing evidence has shown that GPCRs are involved in to B.D.), the National Natural Science Foundation of China (31570896 and 31770969 to viral infection in different manners (7). Additionally, our previous B.D., 81672811 and 81871250 to M.Q., and 81830083 to M.L.), and the Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-05-E00011 to studies also demonstrated that several GPCRs are related to viral M.L.). infection. For example, leucine-rich repeat-containing GPCR 4 B.D., M.Q., Q.W., and M.L. supported and supervised the project. B.D., H.H., and (LGR4) facilitates vesicular stomatitis virus (VSV) infection by N.W. conceived and designed the experiments. N.W., H.H., Q.X., N.C., N.X., and binding VSV glycoprotein (8). Metabolite-sensing GPCR (TGR5), P.W. performed the experiments. B.D., H.H., and N.W. analyzed the data and wrote the manuscript. kisspeptin receptor (GPR54), and purinergic receptors P2Y6 and Address correspondence and reprint requests to Dr. Bing Du or Dr. Qin Wang, P2Y13 modulate the antiviral innate immune responses in different Institute of Biomedical Sciences and School of Life Sciences, East China Normal manners (9–12). Thus, the fundamental roles of these classic G University, 500 Dongchuan Road, Shanghai 200241, China (B.D.) or Department of protein signaling elements are important for us to further understand Nephrology and Rheumatology, Shanghai Fengxian Central Hospital, 6600 Nangfeng Road, Shanghai 201400, China (Q.W.). E-mail addresses: [email protected] the regulation of innate immune responses to virus infection by (B.D.) or [email protected] (Q.W.) GPCRs and extracellular signals. The online version of this article contains supplemental material. Gaq, Gq protein a subunit, encoded by the GNAQ , be- Abbreviations used in this article: 2-APB, 2-aminoethoxydiphenyl borate; BMM, longs to the Gq/11 subfamily of heterotrimeric G , and it is bone marrow–derived macrophage; G protein, guanine nucleotide–binding protein; ubiquitously expressed in mammalian cells (13, 14). GNAQ (Gaq) GPCR, G protein–coupled receptor; HSV-1, HSV type 1; IFN-I, type I IFN; IRF3, IFN regulatory factor 3; MOI, multiplicity of infection; PEM, peritoneal macrophage; initially attracted attention for its physiological significance in the poly(I:C), polyinosinic-polycytidylic acid; Q-PCR, quantitative PCR; RIG-N, N ter- cardiovascular system (15). In recent years, studies have shown minus of RIG-I; siRNA, small interfering RNA; TBK1, TANK-binding kinase 1; the important roles of GNAQ in cancer and autoimmune disease VSV, vesicular stomatitis virus; YM, YM-254890. (16, 17). However, the role of GNAQ in immune responses Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 sometimes seems contrary (17–19). Many GPCRs are found

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900427 2 GNAQ RESTRICTS ANTIVIRAL INNATE IMMUNE RESPONSES coupled with Gaq but play different roles in viral infection. was diluted in 100 ml of serum-free medium, and poly(I:C) (1 mg) was Therefore, it is important for us to explore how Gaq is regulated diluted in 100 ml of serum-free medium for 5 min, respectively. The above by different viruses and GPCRs. In the current study, we dem- two solutions were mixed and incubated at room temperature for 20 min. Subsequently, the mixed solution was added into the cells with 0.8 ml of onstrate that GNAQ is significantly downregulated by different fresh medium. For RNA interference, small interfering RNA (siRNA) viruses and that GNAQ deficiency increases the resistance of the duplexes were transfected using TransExcellent RNA at a ratio of 50 nM host to viral infection both in vitro and in vivo. Consistent with siRNA to 2 ml of TransExcellent RNA in serum-free and antibiotic-free these functional studies, GNAQ-deficient macrophages and mice medium. Medium was changed after 6 h, and experiments were done 48 h b after transfection. Double-stranded siRNA (GenePharma) to silence en- produce more IFN- than wild-type controls. Further results in- dogenous Gnaq expression in PEMs targeted the mouse Gnaq mRNA dicate that GNAQ suppresses IFN-b production through CALNA- sequences 1 (59-GAAGGUGUCUGCUUUUGAGTT-39) and 2 (59-GGA- mediated deactivation of TANK-binding kinase 1 (TBK1) in the GUACAAUCU-GGUCUAAUU-39). Mouse Calna-specific siRNA oligos canonical PLC-b/Ca2+–signaling pathway. Thus, our data suggest (SR416619) were purchased from OriGene. PEMs transfected with an that GNAQ negatively regulates the antiviral innate immune re- equal amount of universal nontargeting siRNA were used as a negative control. sponses and that the downregulation of GNAQ in viral infection may be a novel way for the host to enhance antiviral immunity. Real-time quantitative PCR Cells were lysed, and total RNAwas extracted with TRIzol reagent (Takara). Materials and Methods cDNA was generated using the PrimeScript RT Master Mix Kit (Takara). The cDNA was used as a template and subjected to quantitative PCR (Q-PCR). Mice The primer sequences for Q-PCR analysis are listed in the supplementary Gnaq-knockout mice (Gnaqfl/fl Cre+/2) on a C57BL/6 background were material (Supplemental Table I). kindly provided by J. Luo (East China Normal University). The primers Western blots and immunoprecipitation Downloaded from used for the identification of mutated mice were 59-AGCTTAGT- fl/fl CTGGTGACAGAAGC-39 (Gnaq forward), 59-GCATGCGTGTCCTT- The protein lysates and immunoprecipitations with anti-Flag Ab followed fl/fl TATGTGAG-39 (Gnaq reverse), 59-CTTGGGCTGCCAGAATTTCTC-39 by Western blotting analysis were performed as described previously (10). (Lyz2-Cre common), 59-TTACAGTCGGCCAGGCTGAC-39 (Lyz2-Cre wild- The immunoreactive bands were visualized by the Odyssey system (LI-COR type), and 59-TTACAGTCGGCCAGGCTGAC-39 (Lyz2-Cre mutation). All Biosciences). mice were bred in specific pathogen–free conditions, and all animal experi- ments were approved by the East China Normal University Center for Animal Luciferase reporter assays Research. http://www.jimmunol.org/ HEK-293T cells seeded in 24-well plates were transfected with IFN-b Chemicals and reagents luciferase reporter plasmids together with Renilla plasmids and other de- scribed plasmids for 24 h. Then, the cells were lysed and assayed for DMEM, RPMI 1640, Lipofectamine 2000, and penicillin–streptomycin luciferase activity using the dual-luciferase assay kit (Promega) according were acquired from Invitrogen Life Technologies. FBS was purchased to the manufacturer’s protocol. The data were normalized to the internal from Life Technologies. YM-254890 (YM, GNAQ inhibitor) was Renilla luciferase control. purchased from AdipoGen. U73122 and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Tocris. TransExcellent RNA was provided Lung histology by Y. Cheng (East China Normal University). Polyinosinic-polycytidylic acid [poly(I:C)] was purchased from InvivoGen. M2 beads were purchased Lungs from control or virus-infected mice were dissected, fixed in 4% from Sigma. TRIzol reagent and PrimeScript RT Master Mix were pur- paraformaldehyde, embedded in paraffin, cut into sections, stained with by guest on October 3, 2021 chased from Takara. SYBR Green PCR Master Mix was purchased from H&E solution, and examined by light microscopy for histological changes. Yeasen. The dual-luciferase reporter assay reagent was purchased from Statistical analysis Promega. A mouse IFN-b ELISA kit with precoated plates was purchased from BioLegend. Anti-TBK1, anti–phosphorylated TBK1, anti–IFN reg- Statistical analyses were performed by Student t test using Prism 5.0 ulatory factor 3 (IRF3), anti–phosphorylated IRF3, anti-GNAQ, and anti- (GraphPad Software). Data are presented as the mean 6 SD unless oth- CALNA were purchased from Cell Signaling Technology. Anti-GAPDH, erwise noted, and experiments were repeated at least three times. The anti-Myc and anti-Flag Abs were purchased from Biogot Technology. results were considered significant at p , 0.05. Cell collection and culture Results HEK-293T, L929, and RAW264.7 cells were purchased from the American Type Culture Collection and cultured in complete DMEM containing 10% Viral infection downregulates GNAQ expression FBS and 1% penicillin–streptomycin. Peritoneal macrophages (PEMs) and To explore the potential role of GNAQ in antiviral innate immune bone marrow–derived macrophages (BMMs) were isolated and cultured as previously described (20). regulation, we examined the expression of Gnaq in virus-infected cells. To our surprise, the mRNA expression of Gnaq in mouse Virus propagation PEMs (Fig. 1A), BMMs (Fig. 1B), the mouse fibroblast cell line HSV type 1 (HSV-1) and the Indiana serotype of VSV were provided by L929 (Fig. 1C), and the macrophage-like cell line RAW264.7 P. Wang (Tongji University). VSV-GFP virus was a gift from A. Cimarelli (Fig. 1D) was downregulated by RNA-virus VSV infection. Addi- (Ecole Normale Supe´rieure de Lyon). All viruses were propagated in Vero tionally, similar data were observed in DNA-virus HSV-1–infected cells, and the titers were determined by standard plaque assays. cells (Fig. 1E–H). Furthermore, we also found that the protein ex- Plasmids and transfection pression of GNAQ in PEMs infected with VSV (Fig. 1I) or HSV-1 (Fig. 1J) was reduced. Taken together, these data suggest that viral Flag-GNAQ was cloned into pcDNA3.1. The IFN-b luciferase reporter Renilla, Flag–N terminus of RIG-I (RIG-N), Flag-MAVS, Flag-STING, Flag- infection downregulates GNAQ expression. IRF3 and Flag-TBK1 were kindly provided by P. Wang (Tongji University). GNAQ negatively regulates host defense against viruses Flag-IRF3-5D mutant plasmid and Flag-IRF7 were gifts from D. Xie (Insti- tute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, To delineate the function of GNAQ in host defense against viruses, Chinese Academy of Sciences). Transfections were performed using the we silenced endogenous Gnaq expression in PEMs using calcium phosphate–DNA coprecipitation method for HEK-293T cells, and cells transfected with an equal amount of empty vector were used as controls. Gnaq-specific siRNA (Fig. 2A) and found that knockdown of GNAQ reduced VSV infection (Fig. 2B). This reduction of Viral mimics transfection and RNA interference VSV replication was also observed at different times postinfection PEMs were seeded into 12-well plates at 1 3 106 cells per well. For ac- (Fig. 2C) and VSV multiplicities of infection (MOIs) (Fig. 2D). In tivating cells by transfection with poly(I:C), 2 ml of Lipofectamine 2000 addition to infection with VSV, infection with VSV-GFP was also The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Viral infection downregulates GNAQ expression. (A–D) Q-PCR analysis of Gnaq expression in PEMs (A), BMMs (B), and L929 (C) and RAW264.7 (D) cells infected with RNA virus VSV (MOI of 1) for the indicated times. (E–H) Q-PCR analysis of Gnaq expression in PEMs (E), BMMs (F), and L929 (G) and RAW264.7 (H) cells infected with DNA virus HSV-1 (MOI of 1) for the indicated times. (I and J) Immunoblot analysis of GNAQ protein expression in lysates of PEMs infected with VSV (MOI of 1) (I) and HSV-1 (MOI of 1) (J) for the indicated times. Numbers below lanes indicate grayscale value of GNAQ expression presented relative to GAPDH expression in that same lane. GAPDH was used as an internal control for Q-PCR. Data are representative of three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. by guest on October 3, 2021 reduced in PEMs transfected with Gnaq-specific siRNA (Fig. 2E). i.p. infected wild-type and Gnaq-knockout mice with VSV Consistent with this result, HSV-1 replication was markedly re- (1 3 108 PFU/mouse) for 24 h. VSV replication and titers in the duced in Gnaq-knockdown PEMs (Fig. 2F). Furthermore, we found liver, spleen, and lung were all significantly lower (Fig. 3I, 3J), that VSV replication was decreased significantly by pretreating the and the virus-induced lung tissue injury was ameliorated in Gnaq- PEMs with the GNAQ inhibitor YM (Fig. 2G). At the same time, knockout mice compared with controls (Fig. 3K). Consistent with GNAQ knockdown abolished the inhibition of VSV replication by the reduced VSV replication, the postinfection survival of Gnaq- YM, which confirmed the specific inhibition of GNAQ by YM knockout mice was much greater than that of their wild-type lit- (Fig. 2H). In addition, we transiently expressed GNAQ in HEK- termates (Fig. 3L). Therefore, these results indicate that Gnaq 293T cells (Fig. 2I) and found that overexpression of GNAQ fa- deficiency increases host resistance to viral infection. cilitated VSV and HSV-1 replication in a dose-dependent manner GNAQ negatively regulates IFN-I production (Fig. 2J, 2K). This facilitation of VSV replication by GNAQ was also found at different VSV MOIs (Fig. 2L). Collectively, these Because IFN-I is important for host defense against viruses, we results demonstrate that GNAQ plays a negative role in host defense further examined the expression of IFN-I and found that the ex- against viral infection. pression of IFN-b (both mRNA and protein) was significantly enhanced in Gnaq-knockdown PEMs (Fig. 4A, 4B). Poly(I:C) is GNAQ deficiency increases host resistance to viral infection widely used to mimic RNA virus infection and activate the ex- To further evaluate the importance of GNAQ in viral infection, we tracellular TLR3- or the intracellular RIG-I–associated signaling generated myeloid cell–specific Gnaq-deficient mice. First, we pathways (21, 22). Thus, we transfected or stimulated PEMs with detected the protein expression in PEMs from Gnaqfl/fl (wild-type) poly(I:C) to explore the effect of GNAQ on RIG-I– or TLR3- and Gnaqfl/flCre+/2 (Gnaq-knockout) mice (Fig. 3A). Then, we associated signaling and found that the expression of Ifn-b was challenged PEMs with VSV at different times or MOIs and found enhanced significantly in Gnaq-knockdown PEMs (Fig. 4C, 4D). that VSV replication was significantly reduced in Gnaq-knockout Additionally, we also found that HSV-1–induced Ifn-b production PEMs (Fig. 3B, 3C). Similar data were observed in BMMs was enhanced in Gnaq-knockdown PEMs (Fig. 4E). Similar re- (Fig. 3D, 3E). Moreover, the replication of HSV-1 was also suppressed sults were observed in YM-treated PEMs (Fig. 4F–H). To further in Gnaq-knockout cells (Fig. 3F, 3G). Similarly, VSV and HSV-1 confirm the regulation of GNAQ in IFN-I signaling, we infected titers were lower in the supernatant of Gnaq-knockout PEMs wild-type and Gnaq-knockout PEMs with VSV and found that compared with that of the wild-type cells (Fig. 3H). To further the expression of Ifn-b and IFN-inducible Ifn-a4 was signifi- determine whether GNAQ mediated viral infection in vivo, we cantly enhanced in Gnaq-knockout PEMs (Fig. 4I). As shown in 4 GNAQ RESTRICTS ANTIVIRAL INNATE IMMUNE RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 2. GNAQ negatively regulates host defense against viruses. (A) The mRNA and protein expression of GNAQ in PEMs transfected with Gnaq siRNA for 48 h. (B) Q-PCR analysis of VSV RNA replicates in PEMs (A) infected with VSV (MOI of 1) for 8 h. (C) Q-PCR analysis of VSV RNA replicates in PEMs transfected with Gnaq siRNA for 48 h and then infected with VSV (MOI of 1) for the indicated times. (D) Q-PCR analysis of VSV RNA replicates in PEMs transfected with Gnaq siRNA for 48 h and then infected with the indicated VSV MOI for 8 h. (E) PEMs transfected with Gnaq siRNA for 48 h were infected with VSV-GFP (MOI of 0.01) for 12 h, and VSV-GFP was examined by light microscopy (left panel). The right panel indicates relative fluorescence intensity in the left panel. Original magnification 310. (F) Q-PCR analysis of HSV-1 UL-30 expression in PEMs transfected with Gnaq siRNA for 48 h and then infected with HSV-1 (MOI of 1) for 16 h. (G) Q-PCR analysis of VSV RNA replicates in PEMs pretreated with YM (1 mM) for 1 h and then infected with VSV (MOI of 1) for 12 h. (H) Q-PCR analysis of VSV RNA replicates in PEMs transfected with Gnaq siRNA for 48 h and then pretreated with YM (1 mM) for 1 h and infected with VSV (MOI of 1) for 12 h. (I) The mRNA and protein expression of GNAQ in HEK- 293T cells transfected for 24 h with different amounts (1 and 3 mg) of GNAQ plasmid. (J) Q-PCR analysis of VSV RNA replicates in (I) infected with VSV (MOI of 1) for 8 h. (K) Q-PCR analysis of HSV-1 UL-30 expression in (I) infected with HSV-1 (MOI of 1) for 16 h. (L) Q-PCR analysis of VSV RNA replicates in GNAQ-overexpressing (3 mg) HEK-293T cells infected with the indicated VSV MOI for 12 h. GAPDH was used as an internal control for Q-PCR. Data are representative of three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. BL, bright light; NC, negative control; ns, not significant.

Fig. 4J, VSV-induced IFN-b protein was also enhanced signifi- GNAQ modulates antiviral innate immune responses through cantly in Gnaq-knockout PEMs. Accordingly, the mRNA ex- canonical PLC-b/Ca2+–signaling b pression level of Ifn- was enhanced in poly(I:C)-transfected It is well known that GNAQ directly links receptors to activation (Fig. 4K), poly(I:C)-stimulated (Fig. 4L), or HSV-1–infected of PLC-b isoforms, which, in turn, stimulate inositol lipids and Gnaq-knockout PEMs (Fig. 4M). Furthermore, we also found initiate calcium (Ca2+) mobilization (23). To investigate whether that VSV-induced expression of Ifn-b and IFN-inducible Ifn-a4 this canonical signaling pathway was involved in GNAQ-mediated was significantly enhanced in Gnaq-knockout BMMs (Fig. 4N). antiviral innate immunity, we treated PEMs with U73122 or 2-APB Consistent with the cellular levels, Gnaq-deficient mice produced to block the activation of PLC-b or Ca2+ signaling. U73122 is an much more IFN-b in the serum, liver, spleen, and lung than their N-substituted maleimide that inhibits PLC-b activity through wild-type littermates in response to VSV infection (Fig. 4O, 4P). alkylating cysteine residues of PLC-b (24). 2-APB is a membrane- These consistent results suggest that GNAQ negatively regulates penetrable inhibitor of inositol 1,4,5-trisphosphate (IP3)–induced the production of IFN-I in antiviral innate immune responses both cytosolic Ca2+ release (25). Subsequently, we found that U73122 or in vitro and in vivo. 2-APB did not affect GNAQ expression (Supplemental Fig. 1A, 1B), The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. GNAQ deficiency increases host resistance to viral infection. (A) Immunoblot analysis of GNAQ proteins in lysates of Gnaqfl/fl (wild-type [WT]) and Gnaqfl/fl Cre+/2 (knockout [KO]) PEMs. (B and C) Q-PCR analysis of VSV RNA replicates in WT and KO PEMs infected with VSV (MOI of 1) for the indicated times (B) or with the indicated VSV MOI for 8 h (C). (D and E) Q-PCR analysis of VSV RNA replicates in WT and KO BMMs infected with VSV (MOI of 1) for the indicated times (D) or with the indicated VSV MOI for 8 h (E). (F and G) Q-PCR analysis of HSV-1 UL-30 expression in WT and KO PEMs (F) or BMMs (G) infected with HSV-1 (MOI of 1) for the indicated times. (H) Determination of VSVor HSV-1 titers in supernatant of PEMs by standard plaque assays from WT and KO mice infected with VSV (MOI of 1, 12 h) or HSV-1 (MOI of 1, 24 h). (I) Q-PCR analysis of VSV RNA replicates in the liver, spleen, and lung from WT and KO mice infected with VSV (1 3 108 PFU/mouse) i.p. for 24 h (n = 5; mean 6 SEM). (J) De- termination of VSV loads in the liver, spleen, and lung by standard plaque assays from WT and KO mice i.p. infected with VSV (1 3 108 PFU/mouse) for 24 h. (K) H&E staining of lung sections from mice in (J). Scale bar, 100 mm. (L) Survival of 8-wk-old WT and KO mice given an i.p. injection of VSV (1 3 108 PFU/g) (n =9;mean6 SEM). GAPDH was used as an internal control for Q-PCR. Data are representative of three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. and VSV replication was significantly reduced in U73122- or 2-APB–pretreated Gnaq-knockdown PEMs, suggesting that the 2-APB–treated PEMs in a dose-dependent manner (Fig. 5A). In function of GNAQ is Ca2+-dependent (Fig. 5F). Similar data were addition, the expression of Ifn-b was enhanced in U73122- or 2- also observed in VSV- and HSV-1–infected macrophages (Fig. 5G). APB–treated macrophages (Fig. 5B). Similar results were observed Consistent with IFN-b expression, viral replication was reduced in HSV-1–infected PEMs (Fig. 5C, 5D). Accordingly, we pretreated in Gnaq-knockdown PEMs but not in the 2-APB–pretreated poly(I:C)-stimulated PEMs with U73122 or 2-APB and found that Gnaq-knockdown macrophages (Fig. 5H). Taken together, poly(I:C)-induced expression of Ifn-b was enhanced (Fig. 5E). Most these results suggest that GNAQ modulates antiviral innate importantly, we found that poly(I:C)-stimulated Ifn-b production immune responses through the canonical PLC-b/Ca2+ sig- was significantly enhanced in Gnaq-knockdown PEMs but not in the naling pathway. 6 GNAQ RESTRICTS ANTIVIRAL INNATE IMMUNE RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 4. GNAQ negatively regulates IFN-b production. (A) Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h and then infected with VSV (MOI of 0.5) for 12 h. (B) ELISA of IFN-b levels in supernatants of PEMs transfected with Gnaq siRNA for 48 h and then infectedwithVSV(MOIof0.5)for12h.(C) Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h and then transfected with poly(I:C) (1 mg/ml) for 2 h. (D) Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h and then stimulated with poly(I:C) (10 mg/ml) for 2 h. (E) Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h and then infected with HSV-1 (MOI of 1) for 8 h. (F) Q-PCR analysis of Ifn-b expression in PEMs pretreated with YM (1 mM)for1handtheninfectedwithVSV(MOIof1)for 12 h. (G) Q-PCR analysis of Ifn-b expression in PEMs pretreated with YM (1 mM) for 1 h and then stimulated with poly(I:C) (10 mg/ml) for 2 h. (H) Q-PCR analysis of Ifn-b expression in PEMs pretreated with YM (1 mM) for 1 h and then infected with HSV-1 (MOI of 1) for 8 h. (I) Q-PCR analysis of Ifn-b and Ifn-a4 expression in wild-type (WT) and knockout (KO) PEMs infected with VSV (MOI of 1) for the indicated times. (J) ELISA of IFN- b levels in supernatants of WT and KO PEMs infected with VSV (MOI of 1) for 12 h. (K) Q-PCR analysis of Ifn-b expression in WT and KO PEMs transfected with poly(I:C) (1 mg/ml) for 2 h. (L) Q-PCR analysis of Ifn-b expression in WT and KO PEMs stimulated with poly(I:C) (10 mg/ml) for 2h.(M) Q-PCR analysis of Ifn-b expression in WT and KO PEMs infected with HSV-1 (MOI of 1) for 8 h. (N) Q-PCR analysis of Ifn-b and Ifn-a4 expression in WT and KO BMMs infected with VSV (MOI of 1) for the indicated times. (O) ELISA of IFN-b in sera from WT and KO mice i.p. injected with VSV (1 3 108 PFU/mouse) for 24 h. (P) Q-PCR analysis of Ifn-b expression in the liver, spleen, and lung from mice in (O). GAPDH was used as an internal control for Q-PCR. Data are representative of three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. nd, not detected. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 5. GNAQ modulates antiviral innate immune responses through canonical PLC-b/Ca2+ signaling. (A) Q-PCR analysis of VSV RNA replicates in PEMs pretreated with U73122 or 2-APB for 1 h at the indicated dose (micromolar) and then infected with VSV (MOI of 1) for 8 h. (B) Q-PCR analysis of Ifn-b expression in PEMs pretreated with U73122 (50 mM) or 2-APB (150 mM) for 1 h and then infected with VSV (MOI of 1) for 8 h. (C) Q-PCR analysis of HSV-1 UL-30 expression in PEMs pretreated with U73122 or 2-APB for 1 h at the indicated dose (micromolar) and then infected with HSV-1 (MOI of 1) for 16 h. (D)Q-PCRanalysisofIfn-b expression in PEMs pretreated with U73122 (50 mM) or 2-APB (150 mM) for 1 h and then infected with HSV-1 (MOI of 1) for 8 h. (E) Q-PCR analysis of Ifn-b expression in PEMs pretreated with U73122 (50 mM) or 2-APB (150 mM) for 1 h and then stimulated with poly(I:C) (10 mg/ml) for 2 h. (F) Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h, pretreated with 2-APB (150 mM) for 1 h, and then stimulated with poly(I:C) (10 mg/ml) for 2 h. (G)Q-PCR analysis of Ifn-b expression in PEMs transfected with Gnaq siRNA for 48 h, pretreated with 2-APB (150 mM) for 1 h, and then infected with VSV (MOI of 0.5) for 12 h or HSV-1 (MOI of 1) for 8 h. (H) Q-PCR analysis of VSV RNA replicates or HSV-1 UL-30 expression in PEMs transfected with Gnaq siRNA for 48 h, pretreated with 2-APB (150 mM)for1h,andtheninfectedwithVSV(MOIof1)for12horHSV-1(MOIof1)for16h. GAPDH was used as an internal control for Q-PCR. Data are representative of three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant. 8 GNAQ RESTRICTS ANTIVIRAL INNATE IMMUNE RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 6. The GNAQ/Ca2+/CALNA axis controls IFN-I intensity by dephosphorylating TBK1. (A) Flag-GNAQ was cotransfected with RIG-N, MAVS, STING, TBK1, IKKε, IRF3-5D, IRF7, or vectors, together with an IFN-b luciferase reporter, into HEK-293T cells for 24 h. IFN-b luciferase activity was detected and normalized to Renilla luciferase activity. (B) Different amounts (1 and 3 mg) of Flag-GNAQ were cotransfected with TBK1 or vectors, together with the IFN-b luciferase reporter, into HEK-293T cells for 24 h. IFN-b luciferase activity was detected and normalized to Renilla luciferase activity. (C) Immunoblot analysis of p-TBK1 and p-IRF3 or total proteins in lysates of wild-type (WT) and knockout (KO) PEMs infected with VSV (MOI of 1) for the indicated times. Numbers below lanes indicate grayscale value of p-TBK1 level presented relative to total TBK1 expression level in that same lane. (D) Immunoblot analysis of p-TBK1 or total proteins in lysates of WT and KO PEMs infected with HSV-1 (MOI of 1) for the indicated times. Numbers below lanes indicate grayscale value of p-TBK1 level presented relative to total TBK1 expression level in that same lane. (E) HEK-293T cells were transfected with the indicated plasmids for 24 h and then immunoblotted with the indicated Abs. (F) HEK-293T cells were transfected with plasmid encoding Myc-TBK1 and Flag-CALNA for 24 h. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with Abs to Myc or Flag tags. (G) HEK-293T cells were transfected with the indicated plasmids for 24 h and then immunoblotted with the indicated Abs. (H) Immunoblot analysis of GNAQ and CALNA protein expression in PEMs transfected with Gnaq siRNA and Calna siRNA for 48 h. (I) Q-PCR analysis of Ifn-b expression in (H) stimulated with poly(I:C) (10 mg/ml) for 2 h. (J) Q-PCR analysis of Ifn-b expression in (H) (Figure legend continues) The Journal of Immunology 9

The GNAQ/Ca2+/CALNA axis controls IFN-I intensity by explain why GNAQ deficiency has such significant effects in dephosphorylating TBK1 viral infection, although the closely related GNA11 is still pre- To further investigate the potential mechanism of GNAQ in the sent. In addition, we also demonstrate that the expression of regulation of IFN-I production, we used the IFN-b luciferase re- GNAQ is reduced in viral infection, suggesting that GNAQ- porter to test which components of the pathway are involved in mediated inhibition of antiviral immune responses is restricted GNAQ-mediated regulation. We found that GNAQ significantly by the host immune system to eliminate invading pathogens. Thus, inhibited the RIG-N–, MAVS-, STING-, and TBK1-activated we suggest a negative regulation of GNAQ in antiviral immune IFN-b luciferase activity but did not influence IKKƐ-, IRF3-5D responses, which is involved in the fine-tuning of overactivated (a constitutively active form of IRF3)–, and IRF7-activated IFN-b IFN-I production in viral infection. luciferase activity, suggesting a possible role of TBK1 in GNAQ- In our previous work, we reported that the neuropeptide hormone mediated immune regulation (Fig. 6A). The inhibition of TBK1- kisspeptin can activate GPR54 intracellular signaling to restrict b antiviral innate immune responses (10). Interestingly, GPR54 is a activated IFN- luciferase activity by GNAQ was dose-dependent 2+ (Fig. 6B). Accordingly, the VSV-activated phosphorylation of GNAQ-coupled GPCR and activates intracellular Ca signaling TBK1 (Ser172) and transcription factor IRF3 (Ser396), which are (28). Although we demonstrated that the C terminus of GPR54 responsible for initiating the production of IFN-I, was significantly could bind to calcineurin and that kisspeptin/GPR54 signaling could increase the phosphatase activity of calcineurin to deactivate enhanced in Gnaq-knockout PEMs (Fig. 6C). Similarly, HSV-1– 2+ 172 TBK1 in a Ca -dependent manner, it is not very clear whether activated phosphorylation of TBK1 (Ser ) was also markedly 2+ increased in Gnaq-knockout PEMs (Fig. 6D). In addition, over- GNAQ-activated intracellular Ca is essential for antiviral innate 172 immune responses. In fact, GNAQ is not the only activator of expression of GNAQ inhibited phosphorylation of TBK1 (Ser ) 2+ 2+ Downloaded from (Fig. 6E). Our previous study demonstrated that CALNA binds intracellular Ca , and LPS has been shown to elicit Ca flux in TBK1 and that CALNA phosphatase activity is increased by Ca2+ murine macrophages through a TLR-dependent mechanism that is important for TNF-a production (29, 30). However, TLR- during the activation of kisspeptin/GPR54 signaling (10). We con- 2+ firmed the interaction between CALNA and TBK1 (Fig. 6F) and activated Ca flux has been found to promote IFN-I produc- tion through binding and activating TAK1 and IRF3 by CaMKII that overexpression of CALNA significantly inhibited phosphory- 2+ lation of TBK1 (Ser172) (Fig. 6G). To further investigate the role (Ca /calmodulin-dependent ) (31). These data 2+ http://www.jimmunol.org/ of CALNA in GNAQ regulation of IFN-I production, we cotrans- suggest that intracellular Ca could play a contradictory role in fected Gnaq-specific siRNA and Calna-specific siRNA into PEMs antiviral immune responses by modulating the activity between (Fig. 6H) and found that poly(I:C)-stimulated Ifn-b expression was calcium-dependent protein kinase and phosphatase. In this study, significantly enhanced in Gnaq-knockdown PEMs but not in both we demonstrated that as a negative regulator of antiviral immune Gnaq- and Calna-knockdown PEMs, suggesting that the function of responses, the expression of GNAQ is markedly decreased at both GNAQ is CALNA-dependent (Fig. 6I). Similar data were also ob- the mRNA and protein levels in viral infections, suggesting a served in VSV- or HSV-1–infected macrophages (Fig. 6J). Ac- novel strategy for the host to fight against viral infections. How- cordingly, viral replication was reduced in Gnaq-knockdown PEMs ever, the properties of IFNs have been used to treat different viral infections, inflammatory diseases, and malignancies. Over- but not in both Gnaq-andCalna-knockdown PEMs (Fig. 6K). by guest on October 3, 2021 Taken together, our data indicate that GNAQ limits IFN-I signaling activation of the IFN pathway has been demonstrated in patients through CALNA-mediated deactivation of TBK1. with systemic lupus erythematosus and viral infection–induced lung injury. Thus, immunomodulation strategies designed to de- crease IFN overactivity are also important to avoid IFN-mediated Discussion tissue injury. Thus, our data imply that GNAQ or GNAQ-coupled As an important adapter of GPCRs, GNAQ is ubiquitously GPCRs could be potential drug targets for IFN-mediated immune expressed in mammalian cells and couples a wide variety of re- diseases. ceptors to channel proteins, enzymes, and other effector molecules It is well known that GPCRs couple to several kinds of G protein (23). More remarkably, an increasing number of studies have in- subunits to transduce extracellular signals intracellularly. Thus, it dicated the important roles of GNAQ in regulating both innate and is not surprising that GPCRs could be exploited by viruses to their adaptive immunity (26). For example, GNAQ negatively regulates advantage. GPCRs may help viruses recognize and infect target TCR-mediated immune responses through an LCK-dependent cells or harness their signaling to redirect normal cellular pro- pathway, which suggests a negative regulatory role of GNAQ grams to evade immunodetection or to meet the replicative needs in the transcriptional activation of cytokine responses (27). Sim- of the virus (32). In recent years, the growing list of GPCR sig- ilarly, GNAQ has also been found to reduce the development of naling networks has been found to be hijacked by viral genes in rheumatoid arthritis by inhibiting Th1 and Th17 differentiation viral pathogeneses. In this study, we demonstrated the negative (18, 19). In this study, we provide reliable evidence that GNAQ role of GNAQ in IFN-I–mediated antiviral immune responses negatively regulates antiviral innate immune responses in an through the canonical PLC-b/Ca2+–signaling pathway, implying IFN-I–dependent manner both in vitro and in vivo. It is well that a novel GPCR signaling pathway could be exploited by known that Ga subunits of the Gq class of G proteins include viruses to escape immune defense. Nearly 40% of all GPCRs rely GNAQ, GNA11, GNA14, and GNA15/16 members (23). In some upon GNAQ family members to stimulate Ca2+ signaling (23), cases, the redundancy with GNAQ and GNA11 leads to an ab- implying that GPR54 cannot be the only GPCR hijacked by sence of a phenotype in GNAQ or GNA11 deficiency. However, a virus. Given that GPCRs are the target of almost half of all we found that the expression of GNAQ is much higher than pharmaceutical drugs that are currently available, understanding GNA11 in macrophages (Supplemental Fig. 1C), which may the contribution of GPCR signaling to viral pathogenesis might

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