Portal de Periódicos da CAPES

HDAC 6 regulates cellular viral RNA sensing by deacetylation of RIG ‐I

2016; Springer Nature; Volume: 35; Issue: 4 Linguagem: Inglês

10.15252/embj.201592586

1460-2075

Su Jin Choi, Hyun‐Cheol Lee, Jaehoon Kim, Song Yi Park, Tae‐Hwan Kim, Woon‐Kyu Lee, Duk‐Jae Jang, Ji‐Eun Yoon, Young‐Il Choi, Sei-Hwan Kim, JinYeul Ma, Chul‐Joong Kim, Tso‐Pang Yao, Jae U. Jung, Joo‐Yong Lee, Jong‐Soo Lee,

NF-κB Signaling Pathways

Article8 January 2016free access Source Data HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I Su Jin Choi Su Jin Choi Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Hyun-Cheol Lee Hyun-Cheol Lee College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Jae-Hoon Kim Jae-Hoon Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Song Yi Park Song Yi Park Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Tae-Hwan Kim Tae-Hwan Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Woon-Kyu Lee Woon-Kyu Lee College of Medicine, Inha University, Incheon, Korea Search for more papers by this author Duk-Jae Jang Duk-Jae Jang College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Ji-Eun Yoon Ji-Eun Yoon Foot and Mouth Disease Division, Animal Quarantine and Inspection Agency, Anyang, Korea Search for more papers by this author Young-Il Choi Young-Il Choi CKD Research Institute, Yongin-si, Gyeonggi-do, Korea Search for more papers by this author Seihwan Kim Seihwan Kim CKD Research Institute, Yongin-si, Gyeonggi-do, Korea Search for more papers by this author JinYeul Ma JinYeul Ma Korean Medicine (KM) Based Herbal Drug Development Group, Korea Institute of Oriental Medicine, Daejeon, Korea Search for more papers by this author Chul-Joong Kim Chul-Joong Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Tso-Pang Yao Tso-Pang Yao Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Jae U Jung Jae U Jung Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Joo-Yong Lee Corresponding Author Joo-Yong Lee Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Jong-Soo Lee Corresponding Author Jong-Soo Lee College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Su Jin Choi Su Jin Choi Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Hyun-Cheol Lee Hyun-Cheol Lee College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Jae-Hoon Kim Jae-Hoon Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Song Yi Park Song Yi Park Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Tae-Hwan Kim Tae-Hwan Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Woon-Kyu Lee Woon-Kyu Lee College of Medicine, Inha University, Incheon, Korea Search for more papers by this author Duk-Jae Jang Duk-Jae Jang College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Ji-Eun Yoon Ji-Eun Yoon Foot and Mouth Disease Division, Animal Quarantine and Inspection Agency, Anyang, Korea Search for more papers by this author Young-Il Choi Young-Il Choi CKD Research Institute, Yongin-si, Gyeonggi-do, Korea Search for more papers by this author Seihwan Kim Seihwan Kim CKD Research Institute, Yongin-si, Gyeonggi-do, Korea Search for more papers by this author JinYeul Ma JinYeul Ma Korean Medicine (KM) Based Herbal Drug Development Group, Korea Institute of Oriental Medicine, Daejeon, Korea Search for more papers by this author Chul-Joong Kim Chul-Joong Kim College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Tso-Pang Yao Tso-Pang Yao Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Jae U Jung Jae U Jung Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Joo-Yong Lee Corresponding Author Joo-Yong Lee Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea Search for more papers by this author Jong-Soo Lee Corresponding Author Jong-Soo Lee College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea Search for more papers by this author Author Information Su Jin Choi1,‡, Hyun-Cheol Lee2,‡, Jae-Hoon Kim2, Song Yi Park1, Tae-Hwan Kim2, Woon-Kyu Lee3, Duk-Jae Jang2, Ji-Eun Yoon4, Young-Il Choi5, Seihwan Kim5, JinYeul Ma6, Chul-Joong Kim2, Tso-Pang Yao7, Jae U Jung8, Joo-Yong Lee 1 and Jong-Soo Lee 2 1Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon, Korea 2College of Veterinary Medicine (BK21 Plus Program), Chungnam National University, Daejeon, Korea 3College of Medicine, Inha University, Incheon, Korea 4Foot and Mouth Disease Division, Animal Quarantine and Inspection Agency, Anyang, Korea 5CKD Research Institute, Yongin-si, Gyeonggi-do, Korea 6Korean Medicine (KM) Based Herbal Drug Development Group, Korea Institute of Oriental Medicine, Daejeon, Korea 7Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA 8Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +82 42 821 8559; Fax: +82 42 821 8541; E-mail: [email protected] *Corresponding author. Tel: +82 42 821 6753; Fax: +82 42 821 8903; E-mail: [email protected] The EMBO Journal (2016)35:429-442https://doi.org/10.15252/embj.201592586 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA-sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG-I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG-I-mediated antiviral sensing pathway. Synopsis Deacetylation of the cytoplasmic RIG-I viral sensor by HDAC6 is required for viral RNA detection. HDAC6 depletion impairs in vivo responses against RNA but not DNA viruses in mice. HDAC6 interacts with and deacetylates RIG-I in response to RNA virus infection. Deacetylation of the K909 residue on RIG-I regulates its viral RNA-sensing activity. HDAC6−/− mice are highly susceptible to RNA virus infection. Introduction Retinoic-acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated protein 5 (MDA5), play key roles in innate immune responses against virus infection (Yoneyama et al, 2004, 2005; Akira et al, 2006; Pichlmair & Reis e Sousa, 2007). Invading RNA viruses are recognized by RLRs in the cytosol. RLRs activate the signaling adaptor protein mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, or CARDIF) and initiate downstream signaling pathways to induce antiviral responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines (Akira et al, 2006; Kawai & Akira, 2006; Pichlmair & Reis e Sousa, 2007; Loo & Gale, 2011). RIG-I has been reported to recognize 5′-triphosphate-containing double-stranded RNA from diverse viruses or short double-stranded RNA molecules and has been the subject of extensive research interest as a viral sensor (Yoneyama et al, 2004, 2005; Akira et al, 2006; Pichlmair & Reis e Sousa, 2007). RIG-I comprises two N-terminal tandem caspase activation and recruitment domains (CARDs), a central DExH-box-type RNA helicase composed of two Rec A domains (Hel-1 and Hel-2; the SF2 helicase core), and a C-terminal regulatory domain (CTD) (Wang et al, 2010; Kowalinski et al, 2011). In uninfected cells, RIG-I exists in an autorepressed conformation in which the CARDs are not available for inducing downstream signal transduction (Takahasi et al, 2008). Upon viral infection, viral RNAs bind both the CTD and the helicase domain, thereby activating the ATPase activity of the helicase domain, which in turn triggers a conformational change to expose the masked CARDs (Kowalinski et al, 2011; Luo et al, 2011; Leung & Amarasinghe, 2012). The CARD of RIG-I then interacts with its downstream molecule, MAVS, to trigger antiviral responses (Kowalinski et al, 2011; Luo et al, 2011; Leung & Amarasinghe, 2012). Several positive or negative regulatory mechanisms for the control of RIG-I-mediated antiviral signaling have been described, including posttranslational modifications of RIG-I. The importance of RIG-I ubiquitination (Gack et al, 2007; Oshiumi et al, 2013), phosphorylation (Gack et al, 2010), and SUMOylation (Mi et al, 2010), which regulate its function, has been demonstrated. However, the role of RIG-I acetylation on innate immune responses has not been investigated to date. Regarding the mechanisms of RIG-I-mediated viral sensing, there is still significant uncertainty as to how RIG-I binds and recognizes RNAs in virus-infected cells and how this process is regulated. In addition, the role of RIG-I acetylation in modulating its binding activity or the recognition of viral RNAs remains to be elucidated. Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase important for the regulation of cytoskeletal and mitochondrial functions (Hubbert et al, 2002; Kovacs et al, 2005; Zhang et al, 2007; Lee et al, 2010a,b, 2014). Recently, HDAC6 has been shown to regulate replication of human immunodeficiency virus (HIV) and influenza A virus (IVA) (Valenzuela-Fernandez et al, 2005; Mosley et al, 2006; Husain & Cheung, 2014; Lucera et al, 2014), and innate antiviral immunity (Nusinzon & Horvath, 2006; Zhu et al, 2011; Chattopadhyay et al, 2013). However, it remains unknown how HDAC6 is connected to the antiviral signaling network and whether it plays a critical role in antiviral immunity under physiological conditions in animal. In the present study, we show that HDAC6 knockdown in vitro and HDAC6-deficient mice show impaired innate immune responses against RNA viruses. Investigation of the underlying mechanism showed that, upon RNA virus infection, HDAC6 transiently binds RIG-I and deacetylates lysine 909 (K909), which result in activating CTD of RIG-I to bind and recognize viral RNAs. These findings undercover a novel mechanism of RIG-I activation mediated by HDAC6-dependent deacetylation, facilitating the recognition of viral RNA by the CTD domain of RIG-I to induce innate immune responses against RNA virus infection. Results HDAC6 is essential for the protection of mice against VSV-Indiana infection To determine whether HDAC6 plays a physiological role in an RNA virus infection in vivo, HDAC6+/+ and HDAC6−/− mice were intravenously infected with vesicular stomatitis virus (VSV, Indiana strain). As shown in Fig 1A, HDAC6−/− mice are more susceptible to VSV-Indiana infection than HDAC6+/+ mice and showed significantly decreased survival rate. The virus titer and replication were measured in samples taken from the brain and spleen of mice 5 days after infection with VSV-Indiana; the results showed that viral titers were significantly higher in HDAC6−/− mice than in HDAC6+/+ mice (Fig 1B and C). To determine the effects of HDAC6 deficiency on viral clearance and IFN production in serum, VSV-GFP, which shows low virulence, was intravenously injected into HDAC6+/+ and HDAC6−/− mice and VSV-GFP titers and IFN-β and IL-6 levels in the serum were measured every 6 h. Consistent with previous results, virus titers were significantly higher and IFN-β and IL-6 production was markedly lower in HDAC6−/− mice than in HDAC6+/+ mice (Fig 1D and E). In addition, we examined the role of HDAC6 in cytokine induction by poly(I:C), which is a synthetic double-stranded RNA (dsRNA). Intravenous injection of poly(I:C) caused the rapid and robust induction of IFN-β and IL-6 in HDAC6+/+ mice; however, induction of these cytokines was significantly reduced in HDAC6−/− mice (Fig 1F). These results indicated that HDAC6 plays an important role in the antiviral immune response by producing IFNs and proinflammatory cytokines in responses to foreign RNA viruses. Figure 1. HDAC6−/− mice are susceptible to lethal RNA virus infection and show decreased cytokine production The survival of age- and sex-matched HDAC6+/+ mice and HDAC6−/− mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test). Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6−/− mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann–Whitney test). Determination of viral loads in organs by qPCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6−/− mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann–Whitney test). The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6−/− mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student's t-test). ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (D) (Student's t-test). ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6−/− mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test). Data information: Error bars, mean ± SEM. *P < 0.05, **P < 0.01. Download figure Download PowerPoint HDAC6 is involved in antiviral response against RNA viruses in macrophages and peripheral blood mononuclear cells The antiviral role of HDAC6 was evaluated in bone marrow-derived macrophages (BMDMs) and peripheral blood mononuclear cells (PBMCs) from HDAC6+/+ and HDAC6−/− mice. After infection with VSV-GFP, influenza A virus (PR8-GFP), and Newcastle disease virus (NDV-GFP), virus replication was first measured in HDAC6+/+ and HDAC6−/− BMDMs. As shown in Fig 2A and B and Appendix Fig S1A and B, virus replication was consistently increased in HDAC6−/− BMDMs compared with that in HDAC6+/+ BMDMs. Measurement of IFN-β and IL-6 secretion in BMDMs after virus infection or poly(I:C) and 5′-triphosphate dsRNA (5′ppp-dsRNA) treatment showed that IFN-β and IL-6 production was lower in HDAC6−/− BMDMs than in HDAC6+/+ BMDMs (Fig 2E and Appendix Fig S1C). By contrast, there was no significant difference in virus replication or IFN-β and IL-6 secretion in BMDMs infected by the DNA virus herpes simplex virus (HSV-GFP) or in cells treated with poly(dA:dT), a viral DNA-like molecule (Fig 2C and Appendix Fig S2A). Furthermore, PBMCs isolated from HDAC6+/+ and HDAC6−/− mice infected by VSV-GFP showed a similar pattern to that observed in BMDMs (Fig 2D and Appendix Fig S2B). These results suggested that HDAC6 deficiency suppresses production of type I IFNs and proinflammatory cytokines and selectively enhance the RNA virus infection. Figure 2. HDAC6 positively regulates the innate antiviral response in bone marrow-derived macrophages and peripheral blood mononuclear cells A, B. Virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6−/− BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B). C. Virus replication in HDAC6+/+ and HDAC6−/− BMDMs in response to HSV-GFP (MOI = 2) infection. D. Virus replication in HDAC6+/+ and HDAC6−/− PBMCs in response to VSV-GFP (MOI = 10) infection. E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6−/− BMDMs treated with poly(I:C) (20 μg/ml) or transfected with 5′ppp-dsRNA (1 μg/ml). F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6−/− BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3). G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6−/− BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6−/− BMDMs were stimulated with 5′ppp-dsRNA (0.5 μg/ml) for 6 h. Data information: Data are representative of at least two independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Source data are available online for this figure. Source Data for Figure 2 [embj201592586-sup-0004-SDataFig2F.pdf] Download figure Download PowerPoint We next examined the effects of HDAC6 deficiency on RNA virus-mediated activation of downstream signaling pathways in HDAC6+/+ and HDAC6−/− BMDMs. HDAC6+/+ and HDAC6−/− BMDMs were infected with PR8-GFP, and phosphorylation of IFN-related signaling molecules and phosphorylation of IKBα related to NF-κB activation were confirmed. As shown in Fig 2F and Appendix Fig S3, the phosphorylation levels of IRF3 and TBK1 were markedly lower in HDAC6−/− BMDMs or siHDAC6-transfected RAW264.7 cells than in HDAC6+/+ BMDMs or control RAW264.7 cells, and the levels of phosphorylated IKBα were lower in HDAC6−/− BMDMs than in their HDAC6+/+ counterparts. The mRNA levels of IFN-β, IFN-α, or antiviral-related genes were also assessed in BMDMs with 5′ppp-dsRNA. As shown in Fig 2G, HDAC6−/− BMDMs showed lower levels of gene expression than HDAC6+/+ BMDMs. Collectively, these results suggested that HDAC6 positively regulates the type I IFN signaling pathway and antiviral gene expression induced by RNA virus infection in primary immune cells. HDAC6 positively regulates the innate antiviral response in RAW264.7 cells and fibroblasts The physiological role of HDAC6 in the response to virus infection was further investigated in RAW264.7 cells and in mouse embryonic fibroblasts (MEFs) isolated from HDAC6+/+ or HDAC6−/− mice. We first showed that an HDAC6-specific small interfering RNA (siRNA) efficiently inhibited the expression of endogenous HDAC6 (Appendix Fig S4A). Similar to HDAC6−/− BMDMs or PBMCs, HDAC6 knockdown RAW264.7 cells showed higher levels of virus replication than control cells upon infection with VSV-GFP and PR8-GFP (Fig 3A and B). Consistent with this, we confirmed that siRNA knockdown of HDAC6 significantly reduced the production of IFN-β and IL-6 in RAW264.7 cells infected with virus or in cells treated with poly(I:C) and 5′ppp-dsRNA (Fig 3C). HDAC6-dependent virus replication and induction of IFN-β and IL-6 were also confirmed in fibroblasts (Fig 3D–F and Appendix Fig S3B). Similar to the results obtained in immune cells, HDAC6−/− MEFs showed increased replication of VSV-GFP and PR8-GFP, and lower levels of IFN-β and IL-6 secretion than control cells. Furthermore, when HDAC6−/− MEFs were reconstituted with wild-type HDAC6 (Appendix Fig S3C), the replication of VSV-GFP was reduced (Appendix Fig S3D). Such as the primary immune cell, the mRNA levels of IFN-β, IFN-α, or antiviral-related genes were assessed in HDAC6 knockdown RAW264.7 cells with 5′ppp-dsRNA (Appendix Fig S5) or in HDAC6−/− MEFs with PR8-GFP infection or poly(I:C) transfection (Appendix Fig S6). As shown in figures, HDAC6 knockdown RAW264.7 cells or HDAC6−/− MEFs showed lower levels of gene expression than control Raw 264.7 cells or wild-type cells. Taken together, these results strongly suggested that HDAC6 also plays a role in the antiviral response in immune cell lines and fibroblasts. Figure 3. HDAC6 deficiency reduces the innate immune response on mouse macrophage and mouse embryonic fibroblast A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). Scale bar, 100 μm C. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of cells from (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5′ppp-dsRNA (1 μg/ml). D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6−/− MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). Scale bar, 50 μm. F. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of cells from (D) and (E), and in HDAC6+/+ and HDAC6−/− MEFs transfected with poly(I:C) (1 μg/ml). Data information: Data are representative of at least two independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Download figure Download PowerPoint The catalytic activity of HDAC6 is required for the activation of RIG-I signaling HDAC6 is a well-known cytoplasmic deacetylase. RAW264.7 cells stably expressing HDAC6 or a catalytically inactive HDAC6 mutant (CD mutant; CDM) were used (Appendix Fig S7) to confirm whether the catalytic activity of HDAC6 is required for the antiviral signaling. HDAC6 or HDAC6-CDM stable cells were infected with VSV-GFP and PR8-GFP. We found that virus replication was significantly inhibited in HDAC6-overexpressing RAW264.7 cells; however, virus replication in HDAC6-CDM-overexpressing RAW264.7 cells was similar to that in control cells (Fig 4A and B). Furthermore, PR8-GFP infection was performed after transient reconstitution of HDAC6 and HDAC6-CDM in HDAC6−/− MEFs. Similarly, we found that virus replication was significantly inhibited in HDAC6-overexpressing HDAC6−/− MEFs (Fig EV1). Consistent with this, IFN-β and IL-6 production was significantly higher in HDAC6-overexpressing cells infected with virus or treated with poly(I:C) and 5′ppp-dsRNA than in HDAC6-CDM-overexpressing cells or untreated control cells (Fig 4C), indicating that the catalytic activity of HDAC6 is critical for the antiviral response of HDAC6. Additionally, to confirm the functionality of the catalytic activity of HDAC6, an IFN-β luciferase reporter assay was performed in cells expressing HDAC6 or HDAC6-CDM. HDAC6 significantly enhanced the IFN-β luciferase activity in a dose-dependent manner in response to PR8-GFP infection or treatment with poly(I:C). By contrast, HDAC6-CDM did not enhance PR8-GFP or poly(I:C)-mediated IFN-β luciferase activity (Fig 4D and E). Similar to the virus replication results, these findings indicated that the catalytic activity of HDAC6 is important for the induction of type I IFN signaling. Figure 4. Deacetylase activity is responsible for the antiviral effect of HDAC6 A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector-, HDAC6-, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). Scale bar, 100 μm. C. ELISA of IFN-β (upper) and IL-6 (lower) levels in the supernatant of cells from (A) and (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5′ppp-dsRNA (1 μg/ml). D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h. F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA5 (G), an IFN-β luciferase promoter, and TK-Renilla together with HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay. Data information: Data are representative of three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Catalytic activity of HDAC6 is responsible for antiviral effect HDAC6−/− MEF were transiently transfected with HDAC6-IRES-flag and HDAC6-CDM-IRES-flag. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24 hpi in WT and RIG-I−/− MEFs. Cells were transiently transfected with empty vector, HDAC6, HDAC6-CDM for 36 h, followed by infection with PR8-GFP (MOI = 1). Data are representative of at three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test). Scale bar, 100 μm. Source data are available online for this figure. Download figure Download PowerPoint We next attempted to determine which signaling molecule in the RLR signaling pathway is targeted by HDAC6. Ligand-independent IFN-β induction by RIG-I, MDA5, MAVS, and 2CARD of RIG-I expression was not increased by co-transfection of HDAC6 or HDAC6-CDM in a dose-dependent manner (Fig 4F and G and Appendix Fig S8). These results suggested that although HDAC6 functions as a positive regulator of the RLR signaling pathway during viral infections, a different mechanism must be involved in RIG-I signaling or that upstream of RIG-I, rather than the downstream signaling of the RLR pathway, which is mediated by RIG-I and MAVS interactions. HDAC6 interacts with and deacetylates RIG-I in response to RNA virus infection Several lines of evidence indicate that the cytoplasmic deacetylase, HDAC6, plays a role as a positive regulator of the RLR signaling pathway against RNA virus infection, but is not required against DNA virus infection. In addition, HDAC6 affects RIG-I signaling or signaling upstream of RIG-I. Since RIG-I is listed as an acetylated protein in the acetylome database (Choudhary et al, 2009), we explored the possibility that HDAC6 binds and deacetylates RIG-I upon viral infection. To determine whether HDAC6 interacts with RIG-I, we performed co-immunoprecipitation assays with poly(I:C)-transfected 293T cell lysates using an anti-HDAC6 antibody. As shown in Fig 5A (lane 1), HDAC6 did not interact with RIG-I in the absence of poly(I:C). After 4 h of poly(I:C) transfection, HDAC6 interacted with RIG-I (Fig 5A, lane 2); this interaction was no longer detected after 8 h of transfection (Fig 5A, lane 3). This result suggested that the interaction between HDAC6 and RIG-I is transiently induced by poly(I:C). Figure 5. HDAC6 interacts with and deacetylates RIG-I in response to RNA viral infection 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control. BMDMs were isolated from HDAC6+/+ and HDAC6−/− mice and were transfected with 5′ppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed. HeLa cells were transfected with 5′ppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (scale bar, 20 μm). Arrows indicate colocalization of RIG-I and HDAC6. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24 hpi in RIG-I+/+ and RIG-I−/− MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed by infection with VSV-GFP (MOI = 1). Scale bar, 100 μm. HDAC6+/+, HDAC6−/− MEFs, and HDAC6−/− MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity. Whole cell lysates from HDAC6+/+ and HDAC6−/− MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by Western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensi