ARTICLE

https://doi.org/10.1038/s41467-020-19849-9 OPEN β-arrestin 2 as an activator of cGAS-STING signaling and target of viral immune evasion

Yihua Zhang1,5, Manman Li1,5, Liuyan Li1, Gui Qian1, Yu Wang2, Zijuan Chen1, Jing Liu1, Chao Fang3, Feng Huang4, ✉ Daqiao Guo3, Quanming Zou2, Yiwei Chu1 & Dapeng Yan 1

Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. β-arrestin 2 regulates multiple cellular events through the G protein-

1234567890():,; coupled receptor (GPCR) signaling pathways. Here we demonstrate that β-arrestin 2 also promotes virus-induced production of IFN-β and clearance of viruses in macrophages. β- arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of β-arrestin 2 at Lys171 facilitates the activation of the cGAS–STING signaling and the production of IFN-β. In vitro, viral infection induces the degradation of β-arrestin 2 to facilitate immune evasion, while a β-blocker, carvedilol, rescues β-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of β-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate.

1 Department of Immunology, School of Basic Medical Sciences & Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of MOE/MOH, Fudan University, Shanghai 200032, China. 2 Department of Microbiology and Biochemical Pharmacy, National Engineering Research Centre of Immunological Products, College of Pharmacy, Army Medical University, Chongqing 400038, China. 3 Department of Vascular Surgery, Zhongshan Hospital Affiliated to Fudan University, Institute of Vascular Surgery, Fudan University, Shanghai 200032, China. 4 Beijing Hospital of Traditional Chinese Medicine, ✉ Capital Medical University, Beijing 100010, China. 5These authors contributed equally: Yihua Zhang, Manman Li. email: [email protected]

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

iruses are worldwide issues that threaten human health phosphorylation and degradation of IκB, which suppresses the and lead to huge economic losses and social problems. activation and release of p65, thus inhibiting the activation of NF- V κ 40 β The emergence of the novel coronavirus, SARS-CoV-2, B signaling . Moreover, -arrestin 2 interacts with TRAF6 in has resulted in a tremendous public health crisis in China that is response to LPS or IL-1β stimulation and prevents the oligo- also having an impact at a global level1–3. As the first line of merization and autoubiquitination of TRAF6 to negatively reg- immune response, the innate immunity is activated by recog- ulate the TLR–IL-1R signaling pathways41. The drug carvedilol is nizing pathogen-associated molecular patterns (PAMPs) through used to treat heart failure and hypertension. Mechanistically, a set of pattern recognition receptors (PRRs), which can subse- carvedilol induces β-adrenergic receptor (βAR)-mediated EGFR quently regulate the adaptive immunity and together eliminate transactivation and downstream ERK activation in a β-arrestin- the viruses4. Despite the diversity of viruses, host immune cells dependent manner42. Specifically, carvedilol was found to are able to evolve various powerful PRRs and innate immune increase β-arrestin 2 expression and the recruitment of β-arrestin signaling pathways to detect the PAMPs. Toll-like receptors 2 to the β2AR43,44. (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors In the present study, we observe that β-arrestin 2 positively (RLRs), and DNA sensors recognize viral nucleic acids and regulates the production of IFN-β by targeting cGAS. Meanwhile, initiate downstream signal transduction, resulting in the induc- we also demonstrate that deacetylation of β-arrestin 2 at Lys171 tion of type I interferons (IFNs) and other proinflammatory enhances the antiviral immune response. In addition, carvedilol cytokines to protect the host against virus invasion5,6. The blocks the degradation of β-arrestin 2, which is induced by virus endosomal TLRs recognize viral double-stranded RNA (dsRNA; infection. These findings not only elaborate a novel mechanism of TLR3), single-stranded RNA (ssRNA; TLR7/8), or unmethylated viruses that evade host immune response by degrading the CpG sequences in the DNA (TLR9)7–10. RIG-I and melanoma positive regulator β-arrestin 2 but also demonstrate that carve- differentiation-associated gene 5 (MDA5) are known to recognize dilol can be a potential antiviral drug candidate by promoting β- viral dsRNA11. In addition, DNA sensors, such as cyclic GMP- arrestin 2 expression. AMP synthase (cGAS), recognize dsDNA12–14. TLR3 recruits TIR-domain-containing adapter-inducing interferon-β (TRIF), TNFR-associated factor (TRAF) 2/6, and TANK-binding kinase-1 Results (TBK1)15. RIG-I and MDA5 recruit mitochondrial antiviral- Deficiency of β-arrestin 2 decreases cellular antiviral responses. signaling protein (MAVS) to activate TBK116. cGAS synthesizes To investigate the function of β-arrestin 2 in the antiviral innate cGAMP as a second messenger to activate STING and recruit immune response, we analyzed β-arrestin 2 expression in peri- TBK117–19. The TBK1 complex promotes the phosphorylation of toneal macrophages after infection with herpes simplex virus 1 IFN regulatory factor 3 (IRF3), leading to the dimerization and (HSV-1) or vesicular stomatitis virus (VSV). We observed that β- translocation of IRF3 from the cytoplasm to the nucleus, which arrestin 2 protein level was decreased (Fig. 1a and Supplementary interacts with the interferon-stimulated response elements (ISRE) Fig. 1a), whereas the abundance of mRNA expression of β- to produce type I IFNs and proinflammatory cytokines14. arrestin 2 was not affected by the virus infection (Fig. 1b), indi- cGAS specifically recognizes dsDNA and cDNA that are cating that β-arrestin 2 is probably degraded by viruses in a reverse-transcribed from RNA in RNA viruses, such as HIV-1, to posttranslational manner. produce cGAMP and subsequently activates STING. STING We then prepared peritoneal macrophages from wild-type recruits and activates TBK1 and IRF3, and induces the produc- (WT) and Arrb2−/− mice and stimulated them with HSV-1, VSV, tion of type I IFNs and proinflammatory cytokines. The enzy- Sendai virus (SeV), or DNA mimics. The expression of Ifnb, matic function of cGAS is achieved by the C-terminal part, which Isg15, and Ccl5 was significantly downregulated in Arrb2−/− possesses nucleotidyltransferase activity and MB21 domains macrophages stimulated with all types of stimulations compared along with DNA-binding sites20. The interaction between cGAS with the expression of these genes in their WT counterparts and viral DNA in the cytosol induces the formation of liquid-like (Fig. 1c, d and Supplementary Fig. 1b–e). Consistent with this droplets, resulting in the conformational change and oligomer- finding, the virus titers were much higher in Arrb2−/− ization of cGAS, which promotes the activation of the enzyme macrophages than in WT macrophages (Fig. 1e, f). We next activity of cGAS21,22. Except for the pivotal role of cGAS in virus analyzed the activation of the major pathways that are involved in infection, cGAS is important for maintaining homeostasis in virus infection, including type I IFN, NF-κB, and MAPK several cells. Numerous studies have reported that cGAS could be pathways. We observed that HSV-1 and VSV infection induced regulated by several posttranslational modifications, including reduced phosphorylation of TBK1, IRF3, IRF7, and STING in sumoylation, acetylation, and ubiquitination23–25. On the other Arrb2−/− macrophages than in WT macrophages (Fig. 1g, h). hand, although cGAS acts as a cytosolic DNA sensor that initiates However, the phosphorylation of p65, p38, ERK, and Jnk the STING–TBK1–IRF3-type I IFN signaling cascade, the loca- remained unaffected, indicating that β-arrestin 2 primarily tion of cGAS largely influences its functions. cGAS not only promotes the IFN-β signaling pathway to regulate the antiviral diffuses throughout the cytosol to search its DNA ligand but also innate immune response. The dimerization and the translocation localizes at the plasma membrane and in the nucleus, which is of IRF3 are essential for the production of IFN-β during virus associated with distinguishing self- and viral DNA, DNA damage, infection. As expected, the dimerization (Fig. 1i, j) and and tumor growth26–28. translocation (Fig. 1k, l and Supplementary Fig. 1f) of IRF3 in β-arrestin 2 is a multifunctional adaptor that plays a role in the Arrb2−/− macrophages were significantly reduced compared with desensitization, internalization, and signaling transduction of those in WT macrophages. different types of cell surface receptors, especially activated To further confirm the function of β-arrestin 2, we transfected GPCRs29–33, to regulate signal transduction and participate in RAW264.7 cells with the negative control small interfering RNA different diseases34–37. Previous studies have demonstrated that (siRNA) or β-arrestin 2-specific siRNA and confirmed that only USP33 and Mdm2 function reciprocally and favor respectively β-arrestin 2-specific siRNA decreased β-arrestin 2 expression the stability and lability of the β-arrestin 2 complex38. β-arrestin 2 (Supplementary Fig. 1g, h). As anticipated, HSV-1, VSV, ISD, and prolongs the activation of ERK through the G protein-dependent RNA mimics poly(I:C) stimulated much lower expression of Ifnb, pathway to regulate microglia chemotaxis39. Conversely, the Isg15, and Ccl5 in β-arrestin 2 knockdown cells (Fig. 1m and interaction between β-arrestin 2 and IκB suppresses the Supplementary Fig. 1i–l). Meanwhile, the titers of HSV-1 and

2 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

a b c WT Arrb2 –/– 6 *** 3 *** 2 ns ns

ns ) ) HSV-1 1.5 ns 4 3 ns ns 0 h 4 2 kDa 01481224 (h) mRNA 1 8 h mRNA

β mRNA (fold) 40 -arrestin 2 12 h 2 1 Ifnb Ifnb (fold × 10 0.5 24 h (fold × 10 35 GAPDH *** ** 0 0 Arrb2 0 HSV-1 VSV HSV-1 (h) 0 81624 ISD (h) 0 16

d WT Arrb2 –/– e f 2 25 20 20 ) ** ) ** *** *** 2 3 ) ) 20 2 1.5 *** 4 15 15 15 10 10 mRNA

1 mRNA 10

VSV titer 5 HSV-1 titer 5 Ifnb (fold × 10 0.5 *** Ifnb (fold × 10 5 (PFU/ml × 10 *** ** (PFU/ml × 10 0 0 0 0 –/– –/ – VSV (h) 0 81624 SeV (h)0 8 16 24 WTArrb2 WT Arrb2

ghHSV-1 VSV i HSV-1 –/– –/– WT Arrb2 WT Arrb2 WT Arrb2 –/– kDa 0 1 4 8 1224 0 1 4 8 12 24 (h) kDa 0 1 4 8 12 24 0 1 4 8 12 24 (h) kDa 0481204812(h) 100 70 p-TBK1 70 p-TBK1 Dimer IRF3 70 40 p-IRF3 p-IRF3 40 55 55 p-IRF7 55 p-IRF7 40 Mono IRF3 40 p-STING 40 p-STING 40 IRF3 55 p-p65 55 p-p65 p-p38 35 GAPDH 35 35 p-p38 40 p-Erk p-Erk 55 40 p-Jnk 55 p-Jnk j VSV 55 β β-arrestin 2 –/– -arrestin 2 40 WT Arrb2 35 GAPDH 35 GAPDH kDa 048120 4 812 (h) 100 Dimer IRF3 70 k PBS HSV-1 VSV 55 40 Mono IRF3 WT Arrb2 –/– WT Arrb2 –/– WT Arrb2 –/– 40 IRF3 35 GAPDH DAPI l HSV-1 WT Arrb2 –/– IRF3 kDa 0481204812(h) 40 IRF3 Cyt 70 LaminB1 35 GAPDH IRF3 Merge 40 m Nuc 70

μ LaminB1 GAPDH 13 35

m siControl siβ-arrestin 2 n siControl siβ-arrestin 2 25 3 *** 25 * 5 20 *** ) ) ) )

) ** 2 ** 3

20 4 3 3 10 2 * 4 15 15 *** 15 3 mRNA mRNA mRNA 10 10 1 10 2 Ifnb Ifnb (fold × 10 (fold × 10 Ifnb (fold × 10 5 5 VSV titer HSV-1 titer 1 5 (PFU/ml × 10 0 0 0 (PFU/ml × 10 0 0 HSV-1 (h) 0 816 VSV (h) 0 816 ISD (h) 0 16

o HSV-1 p VSV q HSV-1 siControl siβ-arrestin 2 β siControl si -arrestin 2 siControl siβ-arrestin 2 kDa 0 1 4 8 1224 0 1 4 8 12 24 (h) kDa 0 1 4 8 12 24 0 1 4 8 12 24 (h) kDa 04812 04812(h) p-TBK1 70 70 p-TBK1 p-IRF3 100 Dimer IRF3 40 40 p-IRF3 70 55 p-IRF7 55 p-IRF7 55 40 Mono IRF3 40 p-STING 40 p-STING 55 IRF3 β-arrestin2 55 β-arrestin2 40 35 GAPDH 35 GAPDH 35 GAPDH

VSV were increased in β-arrestin 2 knockdown cells (Fig. 1n). β-arrestin 2 positively regulates IFN-β signaling. Next, we Consistent with the cytokine levels, the knockdown of β-arrestin investigated whether β-arrestin 2 overexpression had a substantial 2 significantly reduced the phosphorylation of TBK1, IRF3, IRF7, effect on IFN-β signaling. We observed that in L929 mouse and STING (Fig. 1o, p) and also the dimerization of IRF3 fibroblast cells, β-arrestin 2 overexpression significantly increased (Fig. 1q) during virus infection. Altogether, these results the HSV-1- or VSV-induced expression of Ifnb, Isg15, and Ccl5 suggested that β-arrestin 2 promoted the IFN-β signaling and (Fig. 2a, b). Similar results were obtained when poly(I:C) or ISD the antiviral immune response induced by RNA or DNA virus. was used as a stimulus (Fig. 2c, d). Consistent with this finding,

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

Fig. 1 Deficiency of β-arrestin 2 decreases cellular antiviral responses. a Immunoblot of lysates of peritoneal macrophages infected with HSV-1 for indicated times. b Arrb2 mRNA levels in peritoneal macrophages infected with HSV-1 or VSV for indicated times. c, d Ifnb mRNA levels in WT or Arrb2−/− mouse peritoneal macrophages stimulated with HSV-1 (left, c) (***P < 0.0001, = 0.0006 in sequence, **P = 0.0029), ISD (right, c) (***P < 0.0001), VSV (left, d) (***P < 0.0001, < 0.0001, < 0.0001 in sequence), or SeV (right, d) (***P = 0.0003, 0.0002 in sequence, **P = 0.0070) for indicated times. e, f The virus titers as in (left, c) (***P = 0.0014) or (left, d) (***P = 0.0031) for 72 h. g, h Immunoblot of lysates of peritoneal macrophages from WT or Arrb2−/− mice infected with HSV-1 g or VSV h for indicated times. i, j Immunoblot analysis of monomeric and dimeric IRF3 as in g or h. k Immunofluorescence microscopy of peritoneal macrophages from WT or Arrb2−/− mice infected with PBS (left), HSV-1 (middle), or VSV (right) for 8h.l Immunoblot analysis of nuclear (Nuc) and cytoplasmic (Cyt) fractions as in i. m Ifnb mRNA levels in RAW264.7 cells transfected with control siRNA or β-arrestin 2 siRNA and then stimulated with HSV-1 (left), VSV (middle), or ISD (right) for indicated times (***P < 0.0001, < 0.0001 in sequence, left panel; ***P < 0.0001, *P = 0.0125, middle panel; *P = 0.0239, right panel). n The virus titers as in m for 72 h (***P = 0.0031, left panel; *P = 0.0019, right panel). o, p Immunoblot of lysates of RAW264.7 cells treated with indicated siRNA and infected with HSV-1 o or VSV p for indicated times. q Immunoblot analysis of monomeric and dimeric IRF3 as in o. ns not significant. Data are representative of at least three independent experiments (mean ± SEM. in b–f, m, n, n = 3). Two-tailed unpaired Student’s t-test.

the titers of HSV-1 or VSV were decreased in β-arrestin 2- defense response to virus (Supplementary Fig. 2c). Gene ontology overexpressing L929 cells (Fig. 2e, f). We next detected the analyses showed that Arrb2−/− group downregulated the phosphorylation of TBK1, IRF3, IRF7, and STING after HSV-1 or expression of multiple genes related to response to external sti- VSV infection. As expected, β-arrestin 2 overexpression in L929 mulus, immune response, inflammatory response, innate immune cells induced even higher phosphorylation of TBK1, IRF3, IRF7, response, response to virus, etc (Fig. 4b and Supplementary and STING than that in the control group (Fig. 2g, h). Further- Fig. 2d). Similarly, KEGG analyses revealed an enrichment of the more, cells with ectopic β-arrestin 2 expression exhibited sig- differential genes in cytokine–cytokine receptor interaction, che- nificantly elevated dimerization of STING (Fig. 2i) and IRF3 mokine signaling pathway, primary immunodeficiency, (Fig. 2j) and translocation of IRF3 to the nucleus (Fig. 2k, l) after Jak–STAT signaling pathway, cytosolic DNA-sensing pathway, stimulation with either HSV-1 or VSV. These results indicated and so on (Fig. 4c and Supplementary Fig. 2e). We next aimed to that β-arrestin 2 enhanced the translocation of IRF3 from the determine the mechanism through which β-arrestin 2 promotes cytoplasm to the nucleus and IFN-β signaling by promoting the IFN-β signaling. To identify the component in IFN-β signaling formation of STING and IRF3 dimers. that was affected by β-arrestin 2, we performed a dual-luciferase assay through overexpressing β-arrestin 2 in HEK293T cells in the presence or absence of expression vectors for RIG-I-N, Deficiency of β-arrestin 2 diminished the antiviral immune MAVS, cGAS plus STING, TBK1, or constitutively active IRF3 response in vivo. To investigate the function of β-arrestin 2 in the (termed as “IRF3-5D” here). Our results revealed that β-arrestin 2 antiviral innate immune response in vivo, we infected 6-week-old overexpression promoted the activity of luciferase reporters for homozygous Arrb2−/− mice and their WT counterparts with above signaling proteins-induced IFN-β and ISRE activation HSV-1 (2 × 107 PFU/mouse) or VSV (5 × 108 PFU/mouse) and except for IRF3-5D (Fig. 4d, e), suggesting that β-arrestin 2 evaluated the expression levels of Ifnb, Isg15, and Ccl5 in different promoted the activation of signaling proteins upstream of IRF3. tissues. After the infection of mice with HSV-1, the expression A co-immunoprecipitation-based screening experiment demon- levels of Ifnb, Ccl5, and Isg15 were significantly lower in the − − strated that β-arrestin 2 interacted with only cGAS (Fig. 4f, g), but spleens, livers, and lungs of Arrb2 / mice than in those of WT not with other proteins in the IFN-β signaling pathway. We next mice (Fig. 3a). Consistent with this result, we observed sig- used E. coli to express and purify GST-cGAS and His-β-arrestin 2. nificantly higher HSV-1 titers in the spleens, livers, and lungs of Using these purified proteins in an in vitro GST precipitation Arrb2−/− mice than in those of WT mice (Fig. 3b). Similar results assay, we found that cGAS directly bound with β-arrestin 2 were obtained when VSV was used as a stimulus (Fig. 3c, d). In (Fig. 4h). The luciferase assay demonstrated that β-arrestin 2 agreement with these results, we observed more injury in the − − promoted cGAS plus STING-triggered activation of the IFNB and lungs and spleens of Arrb2 / mice than in those of WT mice ISRE promoters in a dose-dependent manner (Fig. 4i, j). To (Fig. 3e, f). We further compared the survival rates between WT − − further study which domains of cGAS and β-arrestin 2 are and Arrb2 / mice after infection with HSV-1 or VSV and involved in their interaction, we constructed a series of truncation observed that Arrb2−/− mice were more susceptible to HSV-1 or mutants of cGAS and β-arrestin 2, respectively. We found, using VSV and suffered higher mortality than their WT counterparts truncated proteins, that sequence regions 1–185 of β-arrestin 2 (Fig. 3g, h). All these in vivo results indicated that β-arrestin 2 and 213–522 of cGAS were important for their interactions was a critical positive regulator of the antiviral immune response (Supplementary Fig. 2f, g). to both RNA and DNA viruses. Recognition of dsDNA by cGAS produces cGAMP and subsequently activates STING, which activates IFN-β signaling β-arrestin 2 interacts with cGAS and promotes the activation transduction13,14.Asβ-arrestin 2 was found to interact with of cGAS. To further investigate the function of β-arrestin 2 in cGAS, we assessed the effect of β-arrestin 2 on the binding of host antiviral immunity against virus infection, we performed dsDNA and cGAS. We used a pull-down assay to immunopre- RNA-seq analyses of WT and Arrb2−/− PMs infected with HSV- cipitate biotin-labeled dsDNA with streptavidin and found that β- 1. Compared to WT group, Arrb2−/− group significantly altered arrestin 2 significantly increased the amount of cGAS pulled the expression of 859 genes (251 upregulated and 608 down- down by dsDNA28 (Fig. 4k), indicating that β-arrestin 2 regulated) (Fig. 4a and Supplementary Fig. 2a). The PPI analyses improved the DNA-binding ability of cGAS. We also found that based on differential genes showed a close interaction network β-arrestin 2 could promote cGAS and DNA-binding activity among host antiviral genes, such as Isg15, Cxcl10, Isg20, IFIT in vitro using purified proteins by EMSA (Fig. 4l). As cGAS forms family genes, and so on (Supplementary Fig. 2b). Consistently, dimers after dsDNA binding21,45, we next aimed at investigating functional classification analysis of the downregulated genes whether β-arrestin 2 influenced the formation of cGAS dimers. revealed an enrichment in positive regulation genes of host Our results revealed that β-arrestin 2 overexpression significantly

4 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

a Control β-arrestin 2 100 25 30 ** *** ** ** 80 20 ** 20 60 15 ** *** 40 ** *** 10 mRNA (fold) mRNA (fold)

mRNA (fold) 10 20 5 Ifnb Ccl5 0 lsg15 0 0 HSV-1 (h) 0 81624 HSV-1 (h) 0 81624 HSV-1 (h) 0 81624

b Control β-arrestin 2 40 50 50 *** *** 40 40 30 *** *** 30 ** 30 *** 20 *** 20 20 mRNA (fold) mRNA (fold) mRNA (fold) *** 10 ** 10 10 Ifnb Ccl5

0 lsg15 0 0 VSV (h)0 81624 VSV (h)0 8 16 24 VSV (h) 0 8 16 24

β c Control -arrestin 2 e 10 ** ) ** 2 15 15 4 8 ) ) ) 3 3 *** 3 *** 3 6 10 10 4 mRNA mRNA

mRNA 2

5 5 HSV-1 titer 2 1 × 10 (PFU/ml (fold × 10 (fold × 10 (fold × 10 Ifnb Ccl5

lsg15 0 0 0 0 ISD (h)0 16 ISD (h)0 16 ISD (h) 0 16 Cnotrol β-arrestin 2

d Control β-arrestin 2 f 3 ) 3 ** 15 8 *** 6 ** 2 ) ) ) *** 3 2 3 6 10 4

mRNA 1 mRNA 4 VSV titer mRNA

5 2 × 10 (PFU/ml

(fold × 10 2 (fold × 10 (fold × 10 Ifnb

Ccl5 0 lsg15

0 0 0 trol poly(I:C) (h)0 16 poly(I:C) (h)0 16 poly(I:C) (h) 0 16 Cno β-arrestin 2

g HSV-1 h VSV i Control β-arrestin 2 Control β-arrestin 2 HSV-1

kDa 01481224 01 481224(h) kDa 01 4 81224 0 1 4 8 12 24 (h) Control β-arrestin 2 p-TBK1 70 p-TBK1 70 kDa 048120 4 812(h) p-IRF3 p-IRF3 40 40 100 Dimer STING 55 p-IRF7 55 p-IRF7 70 p-STING 55 40 p-STING 40 40 Mono STING 55 β β-arrestin 2 -arrestin 2 40 40 STING GAPDH 35 GAPDH 35 35 GAPDH

j VSV k HSV-1 l VSV Control β-arrestin 2 Control β-arrestin 2 Control β-arrestin 2 kDa 04 81204812(h) kDa 0481204812(h) kDa 04 812 04812(h) 40 IRF3 40 IRF3 100 Dimer IRF3 Cyt 70 70 LaminB1 Cyt 70 LaminB1 55 35 GAPDH 35 GAPDH 40 Mono IRF3 40 IRF3 40 IRF3 40 IRF3 Nuc 70 LaminB1 Nuc 70 LaminB1 35 GAPDH 35 GAPDH 35 GAPDH

Fig. 2 β-arrestin 2 positively regulates IFN-β signaling. a–d Ifnb, Isg15, and Ccl5 mRNA levels in L929 cells transfected with control or β-arrestin 2 vectors and stimulated with HSV-1 a (**P = 0.0018, ***P < 0.0001, < 0.0001 in sequence, left panel; **P = 0.0040, 0.0028, 0.0050 in sequence, middle panel; ***P = 0.0006, **P = 0.0044, 0.0010 in sequence, right panel), VSV b (***P < 0.0001, **P = 0.0015, 0.0071 in sequence, left panel; ***P < 0.0001, < 0.0001, = 0.0001 in sequence, middle panel; ***P < 0.0001, < 0.0001, = 0.0009 in sequence, right panel), ISD c (***P < 0.0001, left panel; ***P = 0.0004, middle panel; **P = 0.0025, right panel), or poly(I:C) d (***P < 0.0001, left panel; ***P < 0.0001, middle panel; **P = 0.0067, right panel) for indicated times. e, f The virus titers as in a or b for 72 h (**P = 0.0013 in e,**P = 0.0062 in f). g, h Immunoblot of lysates of L929 cells transfected with control or β- arrestin 2 vectors and infected with HSV-1 g or VSV h for indicated times. i, j Immunoblot analysis of monomeric and dimeric STING i and IRF3 j as in g or h. k, l Immunoblot analysis of nuclear and cytoplasmic fractions as in i or j. Data are representative of at least three independent experiments (mean ± SEM in a–f, n = 3). Two-tailed unpaired Student’s t-test.

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

–/– a WT Arrb2 b *** 25 *** 15 5 *** 4 )

*** 5 ) ) )

3 20 2 2 4 3 15 10 3 mRNA

mRNA Spleen 2 Spleen mRNA 10 5 2 1 Ifnb (fold × 10 (fold × 10 (fold × 10 HSV-1 titer

5 Ccl5 1 lsg15 0 0 0 (PFU/ml × 10 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 WTArrb2 –/–

3 1.5 *** 2 15 ***

*** )

*** 3 ) ) ) 3 2 2 1.5 2 1 10

mRNA Liver mRNA 1 Liver mRNA 1 0.5 5 0.5 HSV-1 titer Ifnb (fold × 10 (fold × 10 (fold × 10 Ccl5 Ccl5 lsg15 0 0 0 (PFU/ml × 10 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 WTArrb2 –/–

3 *** 6 4 25 *** *** ) *** 3 ) ) ) 20 3 2 2 3 2 4 15 mRNA

mRNA Lung mRNA 2 Lung 10 1 2 1 HSV-1 titer Ifnb (fold × 10 (fold × 10 (fold × 10

Ccl5 Ccl5 5 lsg15 0 0 0 × 10 (PFU/ml 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 WTArrb2 –/–

c –/– d WT Arrb2 2 1 30 *** 20 ***

*** )

*** 4 ) ) 2 1.5 2 0.8 15 0.6 20 mRNA

mRNA 1 Spleen 10 Spleen 0.4

mRNA (fold) 10 0.5 VSV titer 5 Ifnb (fold × 10 (fold × 10 0.2 lsg15 (PFU/ml × 10 (PFU/ml

0 0 Ccl5 0 0 PBS VSV PBS VSV PBS VSV WTArrb2 –/–

) 25 2.5 *** 2.5 1.5 3 *** *** *** ) ) ) 2 2 2 2 2 20 1.5 1.5 1 15 mRNA mRNA Liver mRNA Liver 10 1 1 0.5 VSV titer Ifnb (fold × 10 (fold × 10 (fold × 10 Ccl5 Ccl5 (PFU/ml × 10 (PFU/ml 0.5 lsg15 0.5 5 0 0 0 0 PBS VSV PBS VSV PBS VSV WTArrb2 –/–

10

1 60 30 ) *** *** *** *** 3

) 8

2 0.8 40 20 0.6 6

Lung mRNA 0.4 Lung 4 mRNA (fold)

20 mRNA (fold) 10 VSV titer Ifnb (fold × 10 0.2 2 (PFU/ml × 10 (PFU/ml

0 Ccl5 0 Isg15 0 0 PBS VSV PBS VSV PBS VSV WTArrb2 –/–

e Lung Spleen f Lung WT Arrb2 –/– WT Arrb2 –/– WT Arrb2 –/–

PBS PBS PBS m m HSV-1 HSV-1 VSV m μ μ μ 147 147 147

g h Spleen WT WT –/– 100 –/– 100 WT Arrb2 Arrb2 Arrb2 –/–

PBS

50 50 * * Survival (%) Survival (%)

0 0

52010 15 51510 20 VSV m μ Day after HSV-1 infection (d) Day after VSV infection (d) 147

Fig. 3 Deficiency of β-arrestin 2 diminished the antiviral immune response in vivo. a Ifnb, Isg15, and Ccl5 mRNA levels in the spleens, livers, and lungs of WT and Arrb2−/− 6-week-old male mice (n = 6 per group) intraperitoneally injected with PBS or HSV-1 (2 × 107 PFU per mouse) for 48 h (***P < 0.0001 in all panels). b The virus titers as in a (***P < 0.0001 in all panels). c VSV (5 × 107 PFU per mouse) infection as in a (***P < 0.0001 in all panels). d The virus titers as in c (***P < 0.0001 in all panels). e, f Microscopy of hematoxylin- and eosin-stained lung and spleen sections as in a or c. g, h Survival of WT and Arrb2−/− 6-week-old male mice (n = 6 per group) infected intraperitoneally with a high dose of HSV-1 (2 × 108 PFU per mouse) g (*P = 0.0430) or VSV (5 × 108 PFU per mouse) h (*P = 0.0411) and monitored for 15 days. Data are representative of at least three independent experiments (mean ± SEM, two- tailed unpaired Student’s t-test in a–d or Kaplan–Meier analysis in g, h).

6 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

a b –log10 (P value) 150 608 251 0 51015 Response to external stimulus Response to stress Immune system process Immune response 100 Defense response Trim29 Adam8 Inflammatory response value) Isg15 Innate immune response P Response to virus Cxcl10 Ccr3 c –log10 (P value)

–log10 ( 50 Mx2 0 2468 Slamf6 Cytokine–cytokine receptor interaction Th17 cell differentiation Chemokine signaling pathway 0 Th1 and Th2 cell differentiation Primary immunodeficiency –5.0 –2.5 0.0 2.55.0 7.5 Toll-like receptor signaling pathway log2 (fold change) Jak-STAT signaling pathway Cytosolic DNA-sensing pathway d e f ) ) 2 2 Control β-arrestin 2 Control β-arrestin 2 2 1 ** *** *** Flag-cGAS + – + 0.8 ** ** 1.5 β ** HA- -arrestin 2 – + + ** 0.6 1 *** β-arrestin 2 0.4 40 ns IP:Flag 70 ns cGAS 0.5 0.2 -Luc activity (fold × 10

β 0 0 Input 40 β-arrestin 2 l kDa IFN

ISRE-Luc activity (fold × 10 G NG -5D tro -5D MAVS TBK1 on MAVS TIN TBK1 Control RIG-I-N IRF3 C RIG-I-N IRF3

cGAS+STI cGAS+S gih Control cGAS+STING 200 T ** GS GST-cGAS 150 β His-β-arrestin 2 + + Flag- -arrestin 2 + – + *** β-arrestin 2 100 *** HA-cGAS – + + 40 cGAS 70 IP:GST 100 50 cGAS -Luc activity (fold) IP:Flag β 0 55 35 GST β-arrestin 2 IFN 0 50 100 200 100 cGAS β Input 70 cGAS Input -arrestin 2(ng) kDa 35 GST kDa

jk l cGAS (mg/ml) – 2 222 Flag-cGAS + +++++ β-arrestin 2 (mg/ml) ––1510 Control cGAS+STING HA-β-arrestin 2 –––+ + + 100 *** 70 cGAS *** Biotin-ISD(h) 001122 80 ** 70 55 60 IP:streptavidin cGAS 40 70 40 cGAS 35 20 β 0 Input 40 -arrestin 2 25 ISRE-Luc activity (fold) 0 50 100 200 kDa Free probe GAPDH β-arrestin 2(ng) 35 kDa

m n p –/– WT Arrb2 –/– WT Arrb2 kDa 048120 4 812 (h) HT-DNA 024 0 2 4 (h) 70 Dimer STING cGAMP 100 55 Flag-cGAS +++ + 70 Dimer IRF3 activity 55 35 Mono STING Mono IRF3 HA-cGAS – + + + assay 40 35 STING HA-β-arrestin 2 ––+ ++ kDa 35 GAPDH 70 HA-cGAS IP:Flag oqsiControl siβ-arrestin 2 β 70 Control -arrestin 2 Flag-cGAS kDa 04812 04 812 (h) kDa 048120 4 812 (h) 70 cGAS 70 Dimer STING 70 Dimer STING Input 55 β-arrestin 2 55 55 kDa 35 Mono STING 40 Mono STING 35 STING 35 35 STING 35 GAPDH 35 GAPDH enhanced the interaction between Flag-tagged cGAS and HA- permeabilized peritoneal macrophages (Fig. 4n), suggesting that tagged cGAS (Fig. 4m). Moreover, we detected the production of β-arrestin 2 deficiency impaired the production of cGAMP. We cGAMP measured by the dimerization of IRF314. In fact, extracts next examined the dimerization of STING and found that from HT-DNA-stimulated Arrb2−/− peritoneal macrophages knockdown or knockout of β-arrestin 2 decreased, but β-arrestin exhibited less cGAMP activity compared with that from WT 2 overexpression increased the dimerization of STING (Fig. 4o, p, macrophages, which resulted in reduced IRF3 dimerization in the q). Taken together, these findings suggested that β-arrestin 2

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

Fig. 4 β-arrestin 2 interacts with cGAS and promotes the activation of cGAS. a Volcano plots comparing gene expression in wild-type versus Arrb2−/− infected peritoneal macrophages. b GO enrichment analysis of differential genes involved in the terms as indicated. c KEGG enrichment analysis of differential genes involved in the pathways as indicated. d, e Luciferase assay of IFN-β d (***P < 0.0001, **P = 0.0033, 0.0021 in sequence, ***P < 0.0001) and ISRE e (**P = 0.0052, ***P = 0.0001, **P = 0.0010, 0.0012 in sequence) activation in HEK293T cells expressing various vectors. f, g Immunoassay of lysates of HEK293T cells expressing various vectors. h Direct binding of His–β-arrestin 2 with GST-cGAS. i, j Luciferase assay of IFN-β i (***P < 0.0001, <0.0001 in sequence, **P = 0.0003) and ISRE j (**P = 0.0001, ***P < 0.0001, < 0.0001 in sequence) activation in HEK293T cells expressing various vectors. k Immunoassay of cell lysates and streptavidin-precipitated proteins from HEK293T cells transfected with various vectors and stimulated with biotin-ISD. l EMSA of cGAS and dsDNA. m Immunoassay of lysates of HEK293T cells expressing various vectors. n cGAMP activity measured by extracts from HT-DNA-stimulated (6 h) WT and Arrb2−/− peritoneal macrophages. o–q Immunoblot analysis of monomeric and dimeric STING in WT and Arrb2−/− peritoneal macrophages o, control siRNA or siβ-arrestin 2-transfected PAW264.7 cells p, and control or β-arrestin 2 vector-transfected L929 cells q.Data are representative of at least three independent experiments (mean ± SEM in d, e, i, j, n = 3). Two-tailed unpaired Student’s t-test.

directly interacted with cGAS and promoted IFN-β signaling by K171R mutant but abrogated by K171Q mutant (Fig. 5i), as well enhancing the DNA-binding ability of cGAS. as the dimerization of cGAS (Fig. 5j), the production of cGAMP As β-arrestin 2 also influenced the VSV-induced activation of (Fig. 5k), and the subsequent dimerization of STING (Fig. 5l), IFN-β signaling, while only binding with cGAS directly, we indicating that the deacetylation of β-arrestin 2 at Lys171 residue thought that there might exist some other proteins that could be was necessary for promoting cGAS activation. Meanwhile, K171R associated with β-arrestin 2. β-arrestin 2 did not associate with mutant further enhanced the interaction between TBK1 and TBK1 or TRAF3 without virus infection, while the situation TRAF3 compared to WT β-arrestin 2 (Supplementary Fig. 3g). changed after infection. We found that endogenous β-arrestin 2 Next, we analyzed the acetylation of β-arrestin 2 during virus could be associated with TBK1 and TRAF3 during virus infection infection and found that β-arrestin 2 was rapidly deacetylated (Supplementary Fig. 2h). Furthermore, β-arrestin 2 could after infection (Fig. 5m and Supplementary Fig. 3h). We also promote the interaction between TBK1 and TRAF3, which might found that ubiquitination of β-arrestin 2 was significantly explain why β-arrestin 2 could promote poly(I:C)- and VSV- increased 12 h after infection, which was coincident with the induced activation of IFN-β signaling (Supplementary Fig. 2i). degradation of β-arrestin 2 (Figs. 1a and 5m and Supplementary Fig. 3h). Consistently, we found that the degradation of β-arrestin Deacetylation of β-arrestin 2 at Lys171 enhanced the antiviral 2 could be rescued by MG-132 after virus infection in peritoneal immune response.Asβ-arrestin 2 expression was decreased after macrophages (Supplementary Fig. 3i, j). Furthermore, the the virus infection, which might be a smart immune evasion acetylation of K171R mutant was significantly decreased and strategy by viruses, we conducted mass spectrometry analysis of not further reduced after the infection, whereas the acetylation of β-arrestin 2 in the presence or absence of HSV-1 infection. This K171Q mutant remained invariable at a high level after the analysis revealed that the acetylation at Lys171 and Lys227 sites infection (Fig. 5n). Consistent with the results that the and the ubiquitination at Lys12, Lys18, Lys171, Lys178, and ubiquitination of both K171R and K171Q mutants was abolished, Lys227 sites were changed after HSV-1 infection. Interestingly, these two mutants were not degraded after the infection (Fig. 5o the Lys171 site of β-arrestin 2 was acetylated before infection but and Supplementary Fig. 3k), which indicated that the ubiquitina- was ubiquitinated after HSV-1 infection (Supplementary Fig. 3a), tion at Lys171 residue induced the degradation of β-arrestin 2. As suggesting that acetylation and ubiquitination regulated the the deacetylation (1 h after infection) of β-arrestin 2 occurred degradation of β-arrestin 2. We replaced the lysines with argi- before the ubiquitination and degradation (12 h after infection) nines (acetylation- and ubiquitination-defective mutants) or during infection, we supposed that Lys171 was deacetylated glutamines (acetylation-mimetic mutants), overexpressed WT β- before being ubiquitinated to positively regulate the IFN-β arrestin 2 and these mutants in HeLa cells, and then infected signaling. them with HSV-1 or VSV to detect the expression of Ifnb.We observed that K171R promoted but K171Q inhibited the Carvedilol promoted the antiviral immunity through β- expression of Ifnb, which indicated that Lys171 is a pivotal resi- arrestin 2. It has been reported that carvedilol could increase due for β-arrestin 2 function (Fig. 5a). Similar results were β-arrestin 2 expression in a rat acute myocardial infarction model β 42–44 obtained with the luciferase assay (Fig. 5b). Furthermore, the and regulate 1AR signaling ; therefore, we speculated whe- production of Ifnb, Isg15, and Ccl5 was increased in Arrb2−/− ther carvedilol could play a role in virus infection through β- mouse embryonic fibroblasts (MEFs) transfected with K171R arrestin 2. We first confirmed the function of carvedilol against mutant compared with WT β-arrestin 2, whereas K171Q mutant virus infection in vivo. The results demonstrated that carvedilol abolished the positive regulation during virus infection (Fig. 5c promoted the production of Ifnb, Isg15, and Ccl5 and reduced the and Supplementary Fig. 3b, c). Consistent with this observation, virus titers in the spleens, livers, and lungs of WT mice after virus K171R mutant inhibited but K171Q mutant promoted the virus infection (Fig. 6a–d). Consistent with this finding, the injury in titers (Fig. 5d). Furthermore, K171R mutant enhanced the the lungs (Fig. 6e) and spleens (Fig. 6f) was less, and the survival phosphorylation of TBK1, IRF3, IRF7, and STING and the rates were higher (Fig. 6g, h) after treatment with carvedilol. In dimerization and translocation of IRF3, compared with WT β- agreement with the in vivo results, the production levels of Ifnb, arrestin 2, whereas K171Q mutant abolished these functions Isg15, and Ccl5 were much higher in the carvedilol-treated WT (Fig. 5e–g, and Supplementary Fig. 3d, e, f). These findings sug- peritoneal macrophages (Fig. 7a, b), whereas the virus titers were gested that the deacetylation of β-arrestin 2 at Lys171 was lower (Fig. 7c, d) after the infection. Furthermore, carvedilol necessary for positive regulation of the IFN-β signaling. increased the phosphorylation of TBK1, IRF3, IRF7, and STING To explore the contribution of Lys171 in β-arrestin 2 to the (Fig. 7e, f) and the dimerization and translocation of IRF3 regulation of cGAS, we detected the interaction between β- (Fig. 7g–j) after the infection. We also detected the production of arrestin 2 mutants and cGAS and found that the interaction was Ifnb, Isg15, and Ccl5 in spleens, livers and lungs in Arrb2−/− enhanced by K171R mutant (Fig. 5h). Meanwhile, we observed mice. We found that carvedilol had no effect on the production of that the DNA-binding ability of cGAS was further enhanced by above cytokines in Arrb2−/− mice after HSV-1 infection

8 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

ab*** *** )

2 Control 8 ** 50 * 2 *** Control WT ** WT 40 *** 6 *** K12R ) *** K12R 2 1.5 30 K18R K18R 4 K171R 1 K171R 20 -Luc activity mRNA (fold) K171Q K171Q β 2 K178R (fold × 10 0.5 mRNA (fold × 10 10 K178R Ifnb K227R IFN K227R

Ifnb 0 0 K227Q 0 K227Q HSV-1 VSV cGAS+STING

cd–/– –/– –/– –/– Arrb2 +Control Arrb2 +WT Arrb2 +K171R Arrb2 +K171Q 30 15 ** *** *** *** 25 20 *** ** *** *** 20 10 20 15 *** ** 15 *** *** 10 10 5 10 mRNA (fold) mRNA (fold) VSV titer (PFU/ml)

5 HSV-1 titer (PFU/ml) 5 0 0 Ifnb Ifnb

0 0 WT WT HSV-1 (h) 0 16 VSV (h) 0 16 Control K171R K171Q Control K171R K171Q

e HSV-1 f HSV-1g HSV-1

Control WT K171R K171Q Control WT K171R K171Q Control WT K171R K171Q kDa 0 8 12 0 8120 812 0 812(h) kDa 0 8 12 0 8 12 0812 0 812 (h) kDa 0808 08 08(h) IRF3 70 p-TBK1 100 40 Dimer IRF3 Cyt 70 LaminB1 p-IRF3 70 40 55 55 p-IRF7 40 Mono IRF3 35 GAPDH IRF3 IRF3 40 p-STING 40 40 LaminB1 55 β-arrestin 2 35 GAPDH Nuc 70 35 GAPDH 35 GAPDH

hi j β HA- -arrestin 2 ––ˉ WT K171RK171Q – HA-β-arrestin 2 – WT K171R K171Q HA-β-arrestin 2 WT K171RK171Q Flag-cGAS + – + + + + Flag-cGAS + + ++ Flag-cGAS + +++ –++ + + + Biotin-ISD(h) +++ + HA-cGAS 55 β -arrestin 2 70 70 HA-cGAS IP:Flag IP:Streptavidin cGAS 70 IP:Flag cGAS 70 70 Flag-cGAS 55 cGAS Input β-arrestin 2 kDa Input 55 β-arrestin 2 55 HA-cGAS Input 55 35 GAPDH β-arrestin2 kDa

k n HSV-1

Control WT K171R K171Q HT-DNA (h) 04040404 HA-β-arrestin2 WT K171R K171Q cGAMP 100 Dimer IRF3 kDa 0 4 0044(h) activity 70 m 55 HSV-1 Ace assay 55 40 Mono IRF3 IP:HA 55 kDa IP:IgG0 148 12 24 (h) β-arrestin 2 IRF3 40 180 55 β GAPDH 130 -arrestin 2 35 Input kDa 100 Ub 35 GAPDH 70 l

-arrestin 2 55 o HSV-1 Control WT K171R K171Q β 55

IP: β-arrestin 2 kDa 0808 0 808 (h) 55 WT K171R K171Q 70 Dimer STING Ace 55 kDa 0 24 0 24 0 24 (h) 55 β-arrestin 2 55 40 Input β-arrestin 2 35 Mono STING 35 GAPDH GAPDH 35 STING 35 35 GAPDH

(Supplementary Fig. 4a). Besides, carvedilol did not increase the As β-arrestin 2 expression was higher in the carvedilol-treated production of Ifnb, Isg15, Ccl5, the phosphorylation of TBK1, group than in the control group 12 h after the infection (Fig. 7e, IRF3, IRF7 and STING in Arrb2−/− peritoneal macrophages f), we hypothesized that β-arrestin 2 degraded by virus infection (Supplementary Fig. 4b–e). These results indicated that carvedilol was rescued by carvedilol to positively regulate the IFN-β promote type I IFN signaling through β-arrestin 2. signaling and the antiviral innate immune response. Furthermore,

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

Fig. 5 Deacetylation of β-arrestin 2 at Lys171 enhanced the antiviral immune response. a Ifnb mRNA level in HeLa cells transfected with control vector, β-arrestin 2, or β-arrestin 2 mutants and infected with HSV-1 (left) or VSV (right) for 16 h (***P < 0.0001, **P = 0.0067, ***P < 0.0001, left panel; ***P = 0.0003, *P = 0.0133, ***P = 0.0003, right panel). b Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors (***P < 0.0001, **P = 0.0017, ***P < 0.0001). c Ifnb mRNA level in Arrb2−/− MEFs transfected with control vector, β-arrestin 2, or β-arrestin 2 mutants and infected with HSV-1 (left) or VSV (right) for 16 h (***P < 0.0001, **P = 0.0032, ***P = 0.0003, left panel; ***P < 0.0001, < 0.0001, = 0.0005 in sequence, right panel). d The virus titers as in c for 72 h (***P = 0.0006, = 0.0003, = 0.0007 in sequence, left panel; ***P < 0.0001, *P = 0.0014, 0.0012 in sequence, right panel). e Immunoassay of lysates of Arrb2−/− MEFs transfected as in c and infected with HSV-1 for indicated times. f Immunoblot analysis of monomeric and dimeric IRF3 as in e. g Immunoblot analysis of nuclear and cytoplasmic fractions as in e. h Immunoassay of lysates of HEK293T cells expressing various vectors. i Immunoassay of cell lysates and streptavidin-precipitated proteins from HEK293T cells transfected with various vectors and stimulated with biotin-ISD. j Immunoassay of lysates of HEK293T cells expressing various vectors. k cGAMP activity measured by extracts from HT-DNA-stimulated (6 h) Arrb2−/− MEFs transfected with various vectors. l Immunoblot analysis of monomeric and dimeric IRF3 as in f. m Immunoassay of lysates of peritoneal macrophages infected with HSV-1 for indicated times. n Immunoassay of lysates of Arrb2−/− MEFs transfected as in l and infected with HSV-1 for 4 h. o Immunoassay of lysates as in n for 24 h. Data are representative of at least three independent experiments (mean ± SEM in b–d, n = 3). Two-tailed unpaired Student’s t-test.

we evaluated the molecular mechanism of carvedilol on the β- which was completely different from the function of β-arrestin 2 arrestin 2-cGAS–STING–IFR3 axis and found that carvedilol in the NF-κB signaling pathway. From the perspective of viruses, significantly enhanced the interaction between β-arrestin 2 and they were so smart that they could inhibit the expression of β- cGAS (Fig. 7k), the DNA-binding ability of cGAS (Fig. 7l), the arrestin 2 by regulating its acetylation and ubiquitination to evade production of cGAMP (Fig. 7m), and the dimerization of STING the host immune response. In addition, except that viruses (Fig. 7n). In consideration of the pivotal role of Lys171, we also inhibited positive regulators, such as β-arrestin 2, to achieve detected the deacetylation of β-arrestin 2 after carvedilol immune evasion in the present study, some other viruses also use treatment and observed that carvedilol promoted the deacetyla- the opposite strategy that enhances the expression of negative tion of β-arrestin 2 during infection (Fig. 7o, p). regulators to eventually evade the host immune response. For To elucidate the relationship between carvedilol and Lys171 of example, one of our previous studies demonstrated that virus- β-arrestin 2, we infected Arrb2−/− MEFs with HSV-1 and VSV, induced PLCβ2 negatively regulates virus-induced proin- which were overexpressed with WT β-arrestin 2 and its mutants, flammatory responses by inhibiting the activation of TAK148. and treated with carvedilol before lysed. The results showed that However, while the devil climbs a post, the priest climbs ten. The carvedilol only enhanced the activity of WT β-arrestin 2 but not clinically proven drug carvedilol rescued the degradation of β- β-arrestin 2 mutants in the production of Ifnb, Isg15, Ccl5, virus arrestin 2 and promoted the activation of IFN-β signaling and the titers, the phosphorylation of TBK1, IRF3, IRF7, STING, the antiviral immune response, indicating that it might be an efficient dimerization, and translocation of IRF3 (Supplementary Fig. 5a- antiviral drug candidate to treat virus infection. g). Furthermore, we detected the interaction between β-arrestin 2 We detected the activation of three major signaling pathways mutants and cGAS, the binding activity between cGAS and in virus infection, including the cGAS–STING, the NF-κB, and dsDNA, the production of cGAMP, and the dimerization of MAPK pathways, and found that β-arrestin 2 promoted only the STING, and found that the function of WT β-arrestin 2, but not activation of the cGAS–STING pathway but had no effect on the β-arrestin 2 mutants, was further promoted by carvedilol other two pathways. In addition, we observed that β-arrestin 2 (Supplementary Fig. 5h-k). Meanwhile, the interaction between enhanced the activation of the cGAS–STING pathway by pro- TBK1 and TRAF3 was promoted not only by carvedilol alone, but moting the recognition of dsDNA by cGAS and the subsequent also through β-arrestin 2 (Supplementary Fig. 5l). And also, the production of cGAMP. However, the detailed mechanism interaction between TBK1 and TRAF3 was further promoted by through which β-arrestin 2 regulates the activation of cGAS and K171R mutant compared to WT β-arrestin 2, while there was no the pivotal residues in cGAS that mediates the activation still need difference with carvedilol treatment (Supplementary Fig. 5m). to be further elucidated. Moreover, no study has yet confirmed Together, we thought that carvedilol activated β-arrestin 2 the enzymatic activity of β-arrestin 2, and it is necessary to fur- through Lys171. These data revealed that carvedilol positively ther elucidate whether β-arrestin 2 directly regulates or recruits regulated IFN-β signaling and antiviral immunity by preventing other enzymes to regulate cGAS activation. the degradation of β-arrestin 2 and promoting the deacetylation We observed that β-arrestin 2 was ubiquitinated and degraded of β-arrestin 2 at Lys171, thus indicating that β-arrestin 2 could 12 h after infection, whereas it was deacetylated only 1 h after be a novel drug candidate for the treatment of virus infection. infection, indicating that β-arrestin 2 could normally promote the activation of IFN-β signaling and the antiviral immune response at the early stage of infection, which was decreased by virus- Discussion induced ubiquitination and degradation of β-arrestin 2 in the late Studies have reported that β-arrestin 2 is widely involved in a stage. However, this type of degradation was not a thorough variety of signaling pathways and functions in multiple diseases, process. In this case, β-arrestin 2 was still expressed at a relatively including nervous and metabolic disorders and several lower level so that it could promote the antiviral immune cancers34,46,47. Furthermore, β-arrestin 2 inhibits the activation of response to some extent. We suppose that this smart and accurate the TRAF6-NF-κB pathway by preventing the self- strategy of immune evasion was developed during the co- oligomerization of TRAF6 or inhibiting the release of p65 from evolution of host and viruses to achieve their symbiosis. This IκB40,41. However, whether and how β-arrestin 2 regulates the mechanism also preserved the antiviral immune response at a host antiviral innate immune response remain unknown. In the relatively lower level but avoided the production of a strong present study, we found that Arrb2−/− mice exhibited lower IFN- cytokine storm, which was another smart strategy for the viruses β production, higher virus titers in tissues, and decreased survival to achieve the symbiosis with the host. Based on the results of rates than their WT littermates. Moreover, β-arrestin 2 promoted mass spectrometry analysis, we assumed that Lys171 is an the activation of the IFN-β signaling pathway by targeting cGAS, important residue as it could be acetylated and ubiquitinated,

10 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

a Control Carvedilol b 4 1.5 1 *** 6 *** *** *** ) 4 ) ) )

2 2 2 0.8 3 4 1 0.6 mRNA mRNA

Spleen mRNA 2 0.4 Spleen 0.5 2

1 HSV-1 titer Ifnb (fold × 10 (fold × 10 (fold × 10

Ccl5 0.2 lsg15 (PFU/ml × 10 0 0 0 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 PBS Carvedilol

2.5 *** 1.5 1.5 5 ) *** 3 ** ) ) )

2 2 2 *** 2 4 1.5 1 1 3

Liver mRNA mRNA mRNA 1 Liver 2 0.5 0.5 HSV-1 titer Ifnb (fold × 10 (fold × 10 (fold × 10

0.5 Ccl5 1 lsg15 (PFU/ml × 10 0 0 0 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 PBS Carvedilol

15 1.5 1.5 25 *** *** ) *** 3

) ) ) ***

2 2 2 20 10 1 1 15 mRNA mRNA Lung mRNA 10 5 0.5 0.5 Lung HSV-1 titer Ifnb (fold × 10 (fold × 10 (fold × 10

Ccl5 5 lsg15 (PFU/ml × 10 0 0 0 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 PBS Carvedilol

c Control Carvedilol d 6 5 4

25 ) *** *** *** 4 *** ) ) )

3 2 20 2 4 3 4 15 3 mRNA

mRNA 2 mRNA Spleen 2 Spleen 2 10 VSV titer 1 Ifnb (fold × 10 (fold × 10 (fold × 10

Ccl5 1

lsg15 5 0 0 0 (PFU/ml × 10 0 PBS VSV PBS VSV PBS VSV PBS Carvedilol

25 8 2.5 *** 8 ***

*** ) *** 3 ) 20 ) )

3 3 2 2 6 6 Liver 15 1.5 mRNA

mRNA 4

mRNA 4 10 1 Liver

2 VSV titer 2 Ifnb (fold × 10 (fold × 10 (fold × 10

5 Ccl5 0.5 lsg15 0 0 0 (PFU/ml × 10 0 PBS VSV PBS VSV PBS VSV PBS Carvedilol

8 8 6 *** 5 *** *** ) *** 3 ) ) ) 4 6 3 6 3 4 4 Lung 3 mRNA

Lung mRNA

mRNA 4 4 2 2 2 2 VSV titer Ifnb (fold × 10 (fold × 10 (fold × 10

Ccl5 1 lsg15 0 0 0 (PFU/ml × 10 0 PBS VSV PBS VSV PBS VSV PBS Carvedilol

e f Lung Lung Spleen Control Carvedilol Control Carvedilol Control Carvedilol

PBS PBS PBS m m VSV m μ HSV-1 HSV-1 μ μ 147 147 147

g h Spleen Control Control Control Carvedilol 100 100 Carvedilol Carvedilol

PBS *

50 * 50 Survival (%) Survival (%)

0 0 VSV m 5 10 15 20 5 10 15 20 μ Day after HSV-1 infection (d) Day after VSV infection (d) 147

which mediated the acetylation and degradation of β-arrestin 2. impaired the activation of IFN-β signaling compared with the We found that β-arrestin 2 was rapidly deacetylated after virus WT β-arrestin 2. We also found that K171R and K171Q were not infection. The acetylation-defective mutant of β-arrestin 2 degraded after virus infection. Thus, the acetylation and ubiqui- (K171R) enhanced but the acetylation-mimetic mutant (K171Q) tination of β-arrestin 2 at Lys171 might mediate the function of

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

Fig. 6 Carvedilol promoted the antiviral immunity in vivo. a Ifnb, Isg15, and Ccl5 mRNA levels in the spleens, livers, and lungs of WT 6-week-old male mice (n = 6 per group) intraperitoneally injected with PBS or HSV-1 (2 × 108 PFU per mouse) for 24 h and then treated with PBS or carvedilol for another 24 h (***P < 0.0001 in all panels). b The virus titers as in a (***P < 0.0001, up panel; **P = 0.0059, middle panel; ***P < 0.0001, below panel). c VSV (5 × 108 PFU per mouse) infection as in a (***P < 0.0001 in all panels). d The virus titers as in c (***P = 0.0005, up panel; ***P = 0.0004, middle panel; ***P = 0.0009, below panel). e, f Microscopy of hematoxylin-and eosin-stained lung and spleen sections as in a or c. g, h Survival of WT 6-week-old male mice (n = 6 per group) infected intraperitoneally with a high dose of HSV-1 (5 × 108 PFU per mouse) g (*P = 0.0485) or VSV (8 × 108 PFU per mouse) (h)(*P = 0.0196) and treated with PBS or carvedilol 24 h after infection and monitored for 15 d. Data are representative of at least three independent experiments (mean ± SEM, two-tailed unpaired Student’s t-test in a–d or Kaplan–Meier analysis in g, h).

β-arrestin 2 in the activation of the IFN-β signaling pathway, the Isolation of mouse peritoneal macrophages and MEFs. For preparing mouse − − specific mechanism of which needs to be further investigated. peritoneal macrophages, 6-week-old homozygous Arrb2 / mice and their WT β littermates were injected with 1.5 ml Brucella broth (4%) intraperitoneally. After In addition, we demonstrated that the -blocker carvedilol fl β 3 days, the peritoneal lavage uid was collected from the mice and washed three rescued the expression of the virus infection-degraded -arrestin times with PBS. Peritoneal macrophages were cultured in DMEM supplemented 2, which further enhanced the antiviral immune response. Car- with 10% FBS. For the preparation of MEFs, 13-day-old embryos of Arrb2−/− mice vedilol also promoted the deacetylation of β-arrestin 2 after virus or their WT littermates were digested with 0.25% trypsin at 4 °C overnight. After infection, which might partially explain why carvedilol could percussion blending, MEFs were washed three times with PBS and then cultured in β DMEM supplemented with 10% (v/v) heat-inactivated FBS (Gibco) and 100 U/ml increase the expression of -arrestin 2 and improve the antiviral penicillin and streptomycin (Hyclone). immune response. These findings revealed that carvedilol might β promote the antiviral innate immune response by enhancing - Virus infection. For in vitro virus infection, L929, HeLa, and RAW264.7 cells and arrestin 2 expression, which may be an outstanding example of peritoneal macrophages (2 × 106 cells) were cultured in DMEM for 12 h and the novel use of a conventional drug to treat viral infectious infected with HSV-1 (MOI = 10) or VSV (MOI = 1) or SeV (100 hemagglutination units [HAU]/ml) for the indicated times. For in vivo virus infection, 6-week-old diseases. − − fi fi β homozygous Arrb2 / mice and their WT littermates were injected with HSV-1 Altogether, our study ndings have identi ed -arrestin 2 as a (2 × 107 PFU/mouse) and VSV (5 × 108 PFU/mouse) for the indicated times. positive regulator of precise control of the cellular antiviral immune response through regulating cGAS activation and IFN-β Transfection. HEK293T cells were transiently transfected with polyethylenimine production. This result should open up new perspectives on the (PEI; 23966-2; Polysciences) according to the manufacturer’s instructions. HeLa or regulation of the cGAS–STING signaling pathway. Furthermore, L929 cells were transfected with Lipofectamine 2000 (11668; Invitrogen), and our study has shed light on the novel mechanism of immune RAW264.7 and MEFs were transfected with Lipofectamine 3000 (L3000; Invitro- evasion by viruses and the possibility for the novel use of the gen). Double-stranded oligonucleotides corresponding to the target sequence (Arrb2, GGAACUCUGUGCGGCUUAUTT) were cloned into the plasmid conventional drug carvedilol as a potential drug candidate for the pLKO.1. treatment of viral infectious diseases. RNA-seq library preparation, sequencing, and data processing.WTandArrb2−/− PMs treated for 16 h with HSV-1 before they were lysed with RNAiso Plus according Methods to the manufacturers’ instructions. The lysates were sent to Personalbio for cDNA Reagents and plasmids. The following chemical reagents and antibodies were library construction and sequencing by Illumina NovaSeq 6000. RNA-sequencing used in this study: anti-TBK1 (3504), anti-phospho-TBK1 (5483), anti-phospho- reads were first trimmed to remove poly(A) and unqualified reads with cutadapt,then IRF3 (29047), anti-phospho-IRF7 (24129), anti-phospho-p65 (3033), anti- aligned to Mus musculus GRCm38 genome with Ensembl. The numbers of counts phospho-p38 (9215), anti-phospho-Erk1/2 (9101), anti-STING (13647), anti-cGAS were summarized at the gene level using HTseq. Gene expression values were com- β (15102), anti-RIG-I (4200), anti-phospho-STING (72971), and anti- -arrestin 2 puted from fragments per kilo bases per million fragments (FPKM) values produced β (3857 S; all from Cell Signaling Technology); anti- -arrestin 2 (ab54790; from by addition of a pseudocount of 1 and log2 transformation of the results. These FPKM fi Abcam) are diluted in the ratio of 1:1000; anti-Flag M2 Af nity Gel (A2220), anti- values were used for drawing the heatmap with the pheatmap R package. Paired HA (H9658), anti-HA (H6908), and anti-Flag (F7425; all from Sigma-Aldrich) are differential gene expression analyses were performed with DEseq R package by diluted in the ratio of 1:10000; anti-GST (CW0085M) and anti-His (CW0083M; addition of fold change > 2 and p-value < 0.05. Volcano plots of these differential both from Cwbio) are diluted in the ratio of 1:10,000; and Protein G Sepharose 4 genes were drawing with ggplots2 R package. GO or KEGG enrichment analyses of Fast Flow (GE Healthcare). Expression constructs for Flag-RIG-I, Flag-RIG-I-N, differential genes were performed using topGO or KAAS, respectively. The terms ɛ Flag-TBK1, Flag-IRF3, Flag-cGAS, Flag-TRAF3, HA-cGAS, HA-CK1 , and HA- or pathways involved in host antiviral immune response with fold change > 2 and Ubs were obtained from Dr. B. Ge (Tongji University, Shanghai, China), and Flag- p-value < 0.05 were used to drawing GO term enrichment or KEGG pathway STING was obtained from Dr. B. Sun (Shanghai Institute of Biochemistry and Cell enrichment. The PPI analysis of differential genes was performed with STRING β Biology, Shanghai, China). Site-directed point mutagenesis of -arrestin 2 was database and PPI network model was drawn with Cytoscape. performed using the KOD Plus Mutagenesis Kit (SMK101, TOYOBO) according to the manufacturer’s instructions. Quantitative real-time PCR. Cells were incubated for 12 h with DMEM and then infected with the viruses for the indicated times. Total RNA was isolated using ’ Cells and viruses RNAiso Plus (9109; Takara) according to the manufacturer s instructions. Then, 1 . Mouse peritoneal macrophages, MEFs, and RAW264.7, L929, ™ and HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium µg of RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (RR037; (DMEM; Hyclone) supplemented with 10% (v/v) heat-inactivated FBS (Gibco) and Takara) to generate cDNA. A LightCycler (LC480; Roche) and a SYBR RT-PCR Kit (QPK-212; Toyobo) were used for quantitative real-time RT-PCR analysis. Gene 100 U/ml penicillin and streptomycin (Hyclone). HeLa cells were cultured in fi ΔΔ RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS (Gibco) ampli cation was performed using the Ct method. Gene expression was nor- and 100 U/ml penicillin and streptomycin (Hyclone). Vero cells were cultured in malized to that of GAPDH. The primer sequences used to amplify human and DMEM supplemented with 3% (v/v) heat-inactivated FBS (Gibco). HSV-1, VSV, mouse genes are described in Supplementary Table 1. and SeV were obtained from Dr. B. Ge (Tongji University, Shanghai, China). Immunoprecipitation and western blotting. Cells were transfected with the indicated plasmids. After 48h, cells were lysed in a lysis buffer (50mM Tris (pH Mouse strains. Homozygous Arrb2−/− mice (from Dr. G. Pei, Tongji University, 7.4), 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA (pH 8.0)) supplemented Shanghai, China) were bred under specific pathogen-free conditions at the with a protease cocktail (Roche, 04693159001) consisting of 1 mM PMSF, 1 mM fi Shanghai Research Center for Biomodel Organisms. Six-week-old male speci c Na3VO4, and1mMNaFfor30minonice.Thelysateswerecentrifugedat pathogen-free (SPF) Arrb2−/− mice and their WT littermates used in the experi- 13,200 rpm for 15 min at 4 °C to remove the debris. Cell lysates were incubated ments were bred separately and put to death by cervical vertebra dislocation. All with anti-Flag M2 Affinity Gel or Protein G Sepharose 4 Fast Flow plus pre- animal studies were approved by the Institutional Animal Care and Use Committee specified antibodies at 4 °C overnight. For immunoprecipitation of endogenous of Fudan University. protein, mouse peritoneal macrophages or HeLa cells were lysed and incubated

12 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

a Control Carvedilol c 15

4 5 3 )

3 ** *** *** *** ) ) 4 ) 4 3 2 2 10 3 2 mRNA mRNA

mRNA 2 2 5 1 HSV-1 titer Ifnb (fold × 10 (fold × 10 1 (fold × 10 Ccl5 (PFU/ml × 10

lsg15 1 0 0 0 0 PBS HSV-1 PBS HSV-1 PBS HSV-1 PBS

Carvedilol

bdControl Carvedilol 4 ) 40 *** 3 ** 25 *** 1.5 3 ) ) ) 3 20 2 *** 2 30 2 15 1 mRNA mRNA

mRNA 20 10 VSV titer 1 0.5 (PFU/ml × 10 Ifnb (fold × 10 10 (fold × 10 (fold × 10 5 Ccl5 lsg15 0 0 0 0 S PBS VSV PBS VSV PBS VSV PB

Carvedilol

e HSV-1 f VSV g HSV-1 Control Carvedilol Control Carvedilol Control Carvedilol kDa 01481224 0 1 4 8 12 24 (h) kDa 01481224 0 1 4 8 12 24 (h) kDa 04812 04812(h) p-TBK1 70 70 p-TBK1 p-IRF3 100 Dimer IRF3 40 40 p-IRF3 70 55 55 p-IRF7 55 p-IRF7 Mono IRF3 40 p-STING IRF3 40 p-STING 40 β-arrestin 2 β-arrestin 2 40 40 35 GAPDH GAPDH 35 35 GAPDH h VSV i HSV-1 j VSV Control Carvedilol Control Carvedilol Control Carvedilol kDa 048120 4 812(h) kDa 04 81204812(h) kDa 0481204812(h) 40 IRF3 40 IRF3 100 Dimer IRF3 Cyt 70 LaminB1 Cyt 70 LaminB1 70 GAPDH GAPDH 55 Mono IRF3 35 35 IRF3 40 40 IRF3 40 IRF3 Nuc 40 70 LaminB1 Nuc 70 LaminB1 35 GAPDH 35 GAPDH 35 GAPDH

k l Biotin-ISD(h) + + + m Control Carvedilol Flag-cGAS +++ HT-DNA 024024(h) Flag-cGAS+ – + + HA-β-arrestin 2 – ++ cGAMP 100 Dimer IRF3 HA-β-arrestin 2 – +++ activity Carvedilol – – + 55 Mono IRF3 Carvedilol –––+ assay kDa 55 cGAS 55 β IP:Streptavidin -arrestin 2 n Control Carvedilol IP:Flag 55 cGAS 70 cGAS kDa 0481204812 (h) Input β-arrestin 2 70 Dimer STING 55 40 Input β-arrestin 2 55 kDa 35 GAPDH 35 Mono STING kDa 35 STING 35 GAPDH

o HSV-1 p VSV Control Carvedilol Control Carvedilol :IgG kDa IP 0 4 8408(h) kDa IP:IgG 0 4 8 084 (h) 55 55 Ace IP: Ace IP: 55 β-arrestin 2 55 β-arrestin 2 β β-arrestin 2 -arrestin 2 55 β 55 β -arrestin 2 -arrestin 2 Input Input GAPDH 35 GAPDH 35

with the specified antibodies and Protein G Sepharose 4 Fast Flow at 4°C GST pull-down. His or GST fusion proteins were expressed in BL-21(DE3) overnight. The sepharose samples were centrifuged and washed three times with (Tiangen Biotech) according to the manufacturer’s instructions. GST fusion pro- ice-cold PBST buffer (1% Triton X-100 in PBS). Precipitates or cell lysates were teins were lysed by ultrasonication, incubated with GST beads for 4 h at 4 °C, and boiled in 1× SDS loading buffer at 100 °C for 10 min and then analyzed by then washed three times with TBST to collect the precipitates. Purified His fusion immunoblotting. protein or lysates of mouse peritoneal macrophages were incubated with the

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9

Fig. 7 Carvedilol promoted cGAS–STING activation through β-arrestin 2. a, b Ifnb, ISG15, and Ccl5 mRNA in WT peritoneal macrophages infected for 16 h with HSV-1 a (***P < 0.0001 in all panels) or VSV b (***P = 0.0002, ***P < 0.0001, ***P < 0.0001) and then treated with PBS or carvedilol. c, d The virus titers as in a or b for 72 h (**P = 0.0058 in c,**P = 0.0050 in d). e, f Immunoblot of lysates of peritoneal macrophages from WT mice infected with HSV-1 e or VSV f for indicated times and treated with PBS or carvedilol. g, h Immunoblot analysis of monomeric and dimeric IRF3 as in e or f. i, j Immunoblot analysis of nuclear and cytoplasmic fractions as in e or f. k Immunoassay of lysates of HEK293T cells transfected with various vectors and treated with PBS or carvedilol. l Immunoassay of cell lysates and streptavidin-precipitated proteins from HEK293T cells transfected with various vectors and stimulated with biotin-ISD and carvedilol. m cGAMP activity measured by extracts from HT-DNA-stimulated (6 h) WT peritoneal macrophages treated with PBS or carvedilol. n Immunoblot analysis of monomeric and dimeric STING as in g. o, p Immunoassay of lysates of peritoneal macrophages infected with HSV-1 o or VSV p for indicated times and treated with PBS or carvedilol. Data are representative of at least three independent experiments (mean ± SEM in a–d, n = 3). **P < 0.01 and ***P < 0.001, two-tailed unpaired Student’s t-test.

collected precipitates for 4 h at 4 °C. After centrifugation and washing, the beads Reporting Summary. Further information on research design is available in were boiled in 1× SDS loading buffer at 100 °C for 10 min and then analyzed by the Nature Research Reporting Summary linked to this article. immunoblotting. Data availability Native PAGE. The dimerization assay was performed as described. In brief, mouse The authors declare that the data supporting the findings of this study are available peritoneal macrophages or L929 cells were cultured in DMEM for 12 h and infected within the paper and its supplementary information files. Source data are provided with with virus for the indicated times. Cells were harvested using 100 μl ice-cold lysis this paper. All other data that support the results of the study will be available from the buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1 mM EDTA, pH 8.0; corresponding author upon reasonable request. The RNA-seq data have been deposited and a protease inhibitor cocktail consisting of 1 mM PMSF, 1 mM Na3VO4, and in Gene Expression Omnibus (GEO) under the GSE159735. Source data are provided 1 mM NaF). After centrifugation at 13,000 × g for 15 min at 4 °C, the supernatants with this paper. were quantified and diluted with 2× native PAGE sample buffer (125 mM Tris- HCl, pH 6.8; 30% glycerol; and 0.1% bromophenol blue). Then, 30 μg of protein was applied to a pre-run 7.5% native gel. After electrophoresis, the proteins were Received: 2 April 2020; Accepted: 3 November 2020; transferred onto a nitrocellulose membrane for immunoblotting.

In vitro assay for cGAMP activity.WTorArrb2−/− mouse peritoneal macro- phages were left untreated or treated with HT-DNA for 6 h and homogenized by douncing in the lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, References 1 mM DTT, and 1 mM PMSF) at 4 °C. The homogenates were centrifuged at 1. The, L. Emerging understandings of 2019-nCoV. Lancet 395, 311 (2020). 10,000 rpm for 20 min at 4 °C. After heating at 95 °C for 5 min, the supernatant was 2. Holshue, M. L. et al. First case of 2019 novel coronavirus in the United States. centrifuged again at 12,000 rpm for 10 min to remove precipitants. The lysates were N. Engl. J. Med. 382, 929–936 (2020). μ mixed with fresh WT mouse peritoneal macrophages in 12.5 l reaction buffer 3. Rothe, C. et al. Transmission of 2019-nCoV infection from an asymptomatic μ μ containing 2 mM ATP, 1 U/ l Benzonase, and 2 ng/ l PFO for 2 or 4 h at 37 °C. contact in Germany. N. Engl. J. Med. 382, 970–971 (2020). The cells were then subjected to the Native PAGE assay. 4. Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature 413, 732–738 (2001). Cell staining and confocal microscopy. HeLa cells were transfected with the 5. Wu, J. & Chen, Z. J. Innate immune sensing and signaling of cytosolic nucleic appropriate plasmids for 48 h and infected with HIV-1 or VSV for the indicated acids. Annu. Rev. Immunol. 32, 461–488 (2014). times. Peritoneal macrophages were infected with viruses directly. Cells were fixed 6. Liuyu, T. et al. Induction of OTUD4 by viral infection promotes antiviral with 4% formaldehyde for 20 min at room temperature, permeabilized for 30 min – in PBS containing 0.3% Triton X-100, and then blocked for 1 h at 4 °C in a blocking responses through deubiquitinating and stabilizing MAVS. Cell Res. 29,67 79 buffer (1% BSA in PBS). Then, the cells were incubated with the indicated anti- (2019). bodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. 7. Zhang, S. et al. Small-molecule inhibition of TLR8 through stabilization of its – After staining with DAPI, images were obtained using a Leica TCS SP5 confocal resting state. Nat. Chem. Biol. 14,58 64 (2018). fi laser microscopy system35. 8. Heil, F. et al. Species-speci c recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529 (2004). 9. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell Dual-luciferase reporter assay. HEK293T cells were transiently transfected with 140, 805–820 (2010). pRL-IFN-β–Luc or pRL-ISRE–Luc, pRL-TK, and the indicated plasmids for 24 h. 10. Jiang, Z., Mak, T. W., Sen, G. & Li, X. Toll-like receptor 3-mediated Dual-luciferase reporter assay system (RG028; Beyotime) was used to detect the activation of NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain- luciferase activity according to the manufacturer’s instructions. containing adapter inducing IFN-beta. Proc. Natl Acad. Sci. USA 101, 3533–3538 (2004). 11. Kato, H. et al. Differential roles of MDA5 and RIG-I helicases in the Electrophoretic mobility shift assay (EMSA). To conduct the EMSA, we firstly recognition of RNA viruses. Nature 441, 101–105 (2006). prepared the lysates of competent E. coli transformed with β-arrestin 2 and cGAS 12. Ablasser, A. & Hur, S. Regulation of cGAS- and RLR-mediated immunity to plasmids for the DNA–protein conjugation reaction and measured the protein nucleic acids. Nat. Immunol. 21,17–29 (2020). concentration by BCA protein assay kit (Beyotime, P0012).We synthesized biotin- 13. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a labeled ISD probe for EMSA assay (Sangon Biotec). Then we executed the cytosolic DNA sensor that activates the type I interferon pathway. Science 339, – DNA protein binding reactions using EMSA Gel-Shift Kit (Beyotime,GS009) fol- 786–791 (2013). ’ lowing the manufacturer s instructions at 25 °C. Finally, the reaction mixture was 14. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate separated through 4% non-denaturing PAGE gels and developed. immune signaling by cytosolic DNA. Science 339, 826–830 (2013). 15. Ye, W. et al. TRIM8 negatively regulates TLR3/4-mediated innate immune response by blocking TRIF-TBK1 interaction. J. Immunol. 199, 1856–1864 Statistical analysis. Data are expressed as mean ± standard error of the mean (2017). (SEM). Prism 6 (GraphPad) is used for statistical analyses. The statistical tests 16. Seth, R. B., Sun, L., Ea, C. K. & Chen, Z. J. Identification and characterization conducted in this study are indicated in the figure legends as follows: *P < 0.05; ** *** ’ of MAVS, a mitochondrial antiviral signaling protein that activates NF- P < 0.01; P < 0.001, two-tailed unpaired Student s t-test. Leica TCS SP8 – confocal laser microscopy system is used for Immunohistochemistry data. ViiA 7 kappaB and IRF 3. Cell 122, 669 682 (2005). Software v1.2.4 on ViiA 7DX is used for qRT-PCR data. ImageQuant LAS 17. Li, X. D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and – 4000mini and Amersham Imager 600 is used for western blot data. RNA-seq data immune adjuvant effects. Science 341, 1390 1394 (2013). are analyzed by cutadapt, Ensembl, HTseq, pheatmap R package, DEseq R, 18. Zhang, C. et al. Structural basis of STING binding with and phosphorylation – ggplots2 R, topGO, KAAS, STRING database, and Cytoscape as described in RNA- by TBK1. Nature 567, 394 398 (2019). seq library preparation, sequencing and data processing section. Illumina NovaSeq 19. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that 6000 is used for RNA sequencing. facilitates innate immune signalling. Nature 455, 674–678 (2008).

14 NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19849-9 ARTICLE

20. Civril, F. et al. Structural mechanism of cytosolic DNA sensing by cGAS. 45. Andreeva, L. et al. cGAS senses long and HMGB/TFAM-bound U-turn DNA Nature 498, 332–337 (2013). by forming protein-DNA ladders. Nature 549, 394–398 (2017). 21. Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS 46. Pydi, S. P. et al. Adipocyte beta-arrestin-2 is essential for maintaining whole activates innate immune signaling. Science 361, 704–709 (2018). body glucose and energy homeostasis. Nat. Commun. 10, 2936 (2019). 22. Xie, W. et al. Human cGAS catalytic domain has an additional DNA-binding 47. Infante, P. et al. Itch/beta-arrestin2-dependent non-proteolytic ubiquitylation interface that enhances enzymatic activity and liquid-phase condensation. of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis. Proc. Natl Acad. Sci. USA 116, 11946–11955 (2019). Nat. Commun. 9, 976 (2018). 23. Hu, M. M. et al. Sumoylation promotes the stability of the DNA sensor cGAS 48. Wang, L. et al. PLCbeta2 negatively regulates the inflammatory response to and the adaptor STING to regulate the kinetics of response to DNA virus. virus infection by inhibiting phosphoinositide-mediated activation of TAK1. Immunity 45, 555–569 (2016). Nat. Commun. 10, 746 (2019). 24. Dai, J. et al. Acetylation blocks cGAS activity and inhibits self-DNA-induced autoimmunity. Cell 176, 1447–1460 (2019). 25. Wang, Q. et al. The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated Acknowledgments − − innate immune response. Plos Pathog. 13, e1006264 (2017). We thank Dr. G. Pei (Tongji University, Shanghai, China) for providing Arrb2 / mice, 26. Barnett, K. C. et al. Phosphoinositide interactions position cGAS at the plasma Dr. B. Ge (Tongji University, Shanghai, China) for providing SeV, VSV, HSV-1, and membrane to ensure efficient distinction between self- and viral DNA. Cell expression constructs for Flag-RIG-I, Flag-N-RIG-I, Flag-TBK1, Flag-IRF3, Flag-cGAS, 176, 1432–1446 (2019). Flag-TRAF3, HA-cGAS, HA-CK1ɛ, and HA-ubiquitinations and Dr. B. Sun for pro- 27. Gentili, M. et al. The N-terminal domain of cGAS determines preferential viding Flag-STING from (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, association with centromeric DNA and innate immune activation in the China). This work was supported by grants from the National Natural Science Foun- nucleus. Cell Rep. 26, 3798 (2019). dation of China (project 31972900 and 31670901), the Program for Professor of Special 28. Liu, H. et al. Nuclear cGAS suppresses DNA repair and promotes Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (program tumorigenesis. Nature 563, 131–136 (2018). TP2016007), the Outstanding Youth Training Program of Shanghai Municipal Com- 29. Chen, W. et al. Activity-dependent internalization of smoothened mediated by mission of Health and Family Planning (program 2017YQ012), Innovative Research beta-arrestin 2 and GRK2. Science 306, 2257–2260 (2004). Team of High-level Local Universities in Shanghai, the National Key Research and 30. Chen, W. et al. Beta-arrestin 2 mediates endocytosis of type III TGF-beta Development Program of China (program 2016YFC1305103 and 2018YFC1705505), and receptor and down-regulation of its signaling. Science 301, 1394–1397 (2003). the National megaproject on key infectious diseases (program 2017ZX10202102). 31. Chen, W. et al. Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A- stimulated endocytosis of Frizzled 4. Science 301, 1391–1394 (2003). Author contributions 32. Luttrell, L. M. & Lefkowitz, R. J. The role of beta-arrestins in the termination Y.Z. and D.Y. designed this study; Y.Z. and M.L. performed the experiments assisted by and transduction of G-protein-coupled receptor signals. J. Cell Sci. 115, L.L., G.Q., Y.W., Z.C., J.L., C.F., and F.H.; D.G., Q.Z., and Y.C. contributed to discussions 455–465 (2002). and agreement with the conclusions; Y.Z. and D.Y. analyzed the data and wrote the 33. Ma, L. & Pei, G. Beta-arrestin signaling and regulation of transcription. J. Cell manuscript; and all authors discussed the results and commented on the manuscript. Sci. 120, 213–218 (2007). 34. Zhu, J. et al. Dopamine D2 receptor restricts astrocytic NLRP3 inflammasome activation via enhancing the interaction of beta-arrestin2 and NLRP3. Cell Competing interests Death Differ. 25, 2037–2049 (2018). The authors declare no competing interests. 35. Infante, P. et al. Itch/beta-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis. Additional information Nat. Commun. 9, 976 (2018). Supplementary information 36. Wang, H. et al. Beta-arrestin2 functions as a key regulator in the sympathetic- is available for this paper at https://doi.org/10.1038/s41467- triggered immunodepression after stroke. J. Neuroinflammation 15, 102 020-19849-9. (2018). Correspondence 37. Walker, J. K. et al. Beta-arrestin-2 regulates the development of allergic and requests for materials should be addressed to D.Y. asthma. J. Clin. Investig. 112, 566–574 (2003). Peer review information 38. Shenoy, S. K. et al. Beta-arrestin-dependent signaling and trafficking of 7- Nature Communications thanks the anonymous reviewer(s) for transmembrane receptors is reciprocally regulated by the deubiquitinase their contribution to the peer review of this work. Peer reviewer reports are available. USP33 and the E3 ligase Mdm2. Proc. Natl Acad. Sci. USA 106, 6650–6655 Reprints and permission information (2009). is available at http://www.nature.com/reprints 39. Lee, S. H., Hollingsworth, R., Kwon, H. Y., Lee, N. & Chung, C. Y. Beta- Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in arrestin 2-dependent activation of ERK1/2 is required for ADP-induced fi paxillin phosphorylation at Ser(83) and microglia chemotaxis. Glia 60, published maps and institutional af liations. 1366–1377 (2012). 40. Gao, H. et al. Identification of beta-arrestin2 as a G protein-coupled receptor- stimulated regulator of NF-kappaB pathways. Mol. Cell 14, 303–317 (2004). Open Access This article is licensed under a Creative Commons 41. Wang, Y. et al. Association of beta-arrestin and TRAF6 negatively regulates Attribution 4.0 International License, which permits use, sharing, toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 7, 139–147 adaptation, distribution and reproduction in any medium or format, as long as you give (2006). appropriate credit to the original author(s) and the source, provide a link to the Creative 42. Kim, I. M. et al. Beta-blockers alprenolol and carvedilol stimulate beta- Commons license, and indicate if changes were made. The images or other third party arrestin-mediated EGFR transactivation. Proc. Natl Acad. Sci. USA 105, material in this article are included in the article’s Creative Commons license, unless 14555–14560 (2008). indicated otherwise in a credit line to the material. If material is not included in the 43. Wisler, J. W. et al. A unique mechanism of beta-blocker action: carvedilol article’s Creative Commons license and your intended use is not permitted by statutory stimulates beta-arrestin signaling. Proc. Natl Acad. Sci. USA 104, 16657–16662 regulation or exceeds the permitted use, you will need to obtain permission directly from (2007). the copyright holder. To view a copy of this license, visit http://creativecommons.org/ 44. Liu, C. C., Huang, Y., Zhang, J. H., Xu, Y. & Wu, C. H. Effect of carvedilol on licenses/by/4.0/. cardiac dysfunction 4 days after myocardial infarction in rats: role of toll-like receptor 4 and beta-arrestin 2. Eur. Rev. Med. Pharm. Sci. 17, 2103–2110 (2013). © The Author(s) 2020

NATURE COMMUNICATIONS | (2020) 11:6000 | https://doi.org/10.1038/s41467-020-19849-9 | www.nature.com/naturecommunications 15