The Journal of Immunology

The Hepatitis B X Protein Disrupts Innate Immunity by Downregulating Mitochondrial Antiviral Signaling Protein

Congwen Wei,*,1 Caifei Ni,*,1 Ting Song,*,1 Yu Liu,† XiaoLi Yang,† Zirui Zheng,* Yongxia Jia,* Yuan Yuan,* Kai Guan,* Yang Xu,‡ Xiaozhong Cheng,* Yanhong Zhang,* Xiao Yang,* Youliang Wang,* Chaoyang Wen,x Qing Wu,x Wei Shi,‡ and Hui Zhong*

Previous studies have shown that both hepatitis Avirus and hepatitis C virus inhibit innate immunity by cleaving the mitochondrial antiviral signaling (MAVS) protein, an essential component of the virus-activated signaling pathway that activates NF-kB and IFN regulatory factor-3 to induce the production of type I IFN. For human hepatitis B virus (HBV), hepatitis B s-Ag, hepatitis B e-Ag, or HBV virions have been shown to suppress TLR-induced antiviral activity with reduced IFN-b production and subsequent induction of IFN-stimulated . However, HBV-mediated suppression of the RIG-I–MDA5 pathway is unknown. In this study, we found that HBV suppressed poly(deoxyadenylate-thymidylate)-activated IFN-b production in hepatocytes. Specifically, hepatitis B virus X (HBX) interacted with MAVS and promoted the degradation of MAVS through Lys136 ubiquitin in MAVS protein, thus preventing the induction of IFN-b. Further analysis of clinical samples revealed that MAVS protein was downregulated in hepatocellular carcinomas of HBV origin, which correlated with increased sensitivities of primary murine hepatocytes isolated from HBX knock-in transgenic mice upon vesicular stomatitis virus infections. By establishing a link between MAVS and HBX, this study suggests that HBV can target the RIG-I signaling by HBX-mediated MAVS downregulation, thereby attenuating the antiviral response of the . The Journal of Immunology, 2010, 185: 1158–1168.

epatitis B virus (HBV), a member of the Hepadnaviridae (2, 3) and cytosolic sensory molecules, such as the multidomain- family, is a hepatotropic noncytopathic DNA virus that is containing NOD proteins, RIG-I, and MDA5 (4–6). Both H estimated to infect 300 million people, with a particularly RIG-I and MDA5 contain caspase recruitment domains (CARDs) high prevalence found in Asia and Africa. Approximately 5% of the that interact with the CARD domain-containing protein mitochon- infected individuals develop chronic infection. Most chronically in- drial antiviral signaling (MAVS) upon binding to uncapped RNA, fected patients remain largely asymptomatic without life-threatening resulting in MAVS association with IkB kinase (IKK) proteins. liver disease, but 10–30% of the patients develop liver cirrhosis with MAVS association with IKKa/b activates NF-kB; its association possible progression to liver cancer. Mechanisms involved in viral with TBK1 as well as IKKε leads to activation of IFN regulatory clearance and persistence remain elusive (1). factor (IRF)-3. Coordinated activation of NF-kB and IRF-3 path- The innate immune system, which is characterized by production ways leads to the assembly of a multiprotein enhancer complex that of type I IFN-a/b cytokines and activation of NK cells, plays drives expression of IFN-b–and IFN-mediated antiviral immunity important roles in the detection and elimination of invading (7–11). As a countermeasure, many have evolved strategies pathogens. The host senses viral and bacterial pathogen invasion to interfere with these innate signaling events and thus inhibit by recognition of pathogen-associated molecular patterns with IFN-b production. For example, hepatits C virus (HCV) NS3/4A pattern recognition receptors, including membrane-bound TLRs protease blocks the RIG-I–mediated signaling pathway by cleaving the MAVS protein to block IFN-b expression (12–17). In a remarkable parallel to the HCV NS3/4A protease, a stable inter- *Beijing Institute of Biotechnology and †The General Hospital of Chinese People’s Armed Police Forces; xChinese People’s Liberation Army General Hospital, Beijing; mediate product of hepatitis A virus (HAV) polyprotein processing, and ‡Key Laboratory for Molecular Enzymology and Engineering, JiLin University, 3ABC, also targets MAVS for proteolysis (18). Changchun, China For HBV, however, little is known about the role of the innate 1 C.W., C.N., and T.S. contributed equally to this work. immune system in HBV infection. Wu et al. (19) demonstrated that Received for publication December 8, 2009. Accepted for publication May 16, 2010. activation of the local innate immune system of the liver through This work was supported in part by National Natural Science Foundation of China the TLR system was able to effectively suppress HBV replication Grants 30772605, 30700413, 30870500, and 30871276 and in part by Beijing Natural in vivo and in vitro. They also showed that hepatitis B s-Ag (HBsAg), Science Foundation Grant 7092081. hepatitis B e-Ag (HBeAg), or HBV virions almost completely Address correspondence and reprint requests to Drs. Hui Zhong and Wei Shi, Beijing Institute of Biotechnology, Beijing, China. E-mail addresses: [email protected] and abrogated TLR-induced antiviral activity, which correlated with sup- [email protected] pression of IFN-b production and subsequent induction of IFN- The online version of this article contains supplemental material. stimulated genes as well as suppressed activation of IRF-3, NF-kB, Abbreviations used in this paper: CARD, caspase recruitment domain; HA, hemagglu- and ERK1/2 (20). These studies indicate that HBV can target the tinin; HAV, hepatitis A virus; HBeAg, hepatitis B e-Ag; HBsAg, hepatitis B s-Ag; HBV, TLR system and thereby attenuate the antiviral response of the innate hepatitis B virus; HBX, hepatitis B virus X; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IB, immunoblotting; IKK, IkB kinase; IRF, IFN regulatory factor; immune system. However, the role of the intracellular RIG-I–MAD5 MAVS, mitochondrial antiviral signaling; N–RIG-I, RIG-I N-terminal CARD-like do- innate immune system in HBV infection remains elusive. Recent main mutant; poly(dAT:dAT), poly(deoxyadenylate-thymidylate); TM, transmembrane; studies have shown that primary hepatocytes retain robust IFN-b VSV, vesicular stomatitis virus; WT, wild-type. responses to intracellular polyinosinic:polycytidylic acid as well Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 as to Sendai virus infection (21). Additionally, activation of IFN www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903874 The Journal of Immunology 1159 expression by cytosolic dsDNA also requires the intracellular dsRNA Real-time PCR was performed using QuantiTect SYBR Green PCR Master sensor RIG-I and its adaptor molecule MAVS (22, 23), raising the Mix (Qiagen, Valencia, CA) in triplicate experiments and analyzed on an ABI possibility that HBV might have also evolved strategies to inter- Prism 7700 analyzer (Applied Biosystems, Foster City, CA). All real-time values were normalized to 18S rRNAwith IFN-b using the following primers: rupt the intracellular RIG-I–MAVS signaling pathway. IFN-b sense, 59-CACGACAGCTCTTTCCATGA-39;IFN-b antisense, 59- Among the proteins encoded by HBV, the hepatitis B virus X AGCCAGTGCTCGATGAATCT-39. (HBX) protein is essential for viral replication in vivo (24) and is In vivo ubiquitination assays thought to contribute to hepatocarcinogenesis (25–27). HBX exerts most of its activities through direct interaction with TATA- HEK293 cells were cotransfected with plasmids expressing Flag-MAVS, Myc-HBX, and hemagglutinin (HA)-tagged ubiquitin. Cells were treated binding proteins, leucine-zipper proteins, and DNA repair proteins with MG132 (20 mM, 6 h) at 48 h after transfection, and they were then (28–32). In this study, we report that MAVS interacts with HBX immunoprecipitated with anti-Flag Ab. and is another new target of HBX protein. We found that HBX Clinical samples promoted the degradation of MAVS, thus preventing the induction of IFN-b. Further analysis of clinical samples revealed that MAVS Liver tumor samples and adjacent noncancerous tissues were obtained from protein was downregulated in hepatocellular carcinomas (HCCs) Shaanxi Chao Ying Biotechnology (Xi’an, Shannxi, China). Immunohisto- chemistry was performed as previously described (35). Rabbit anti-MAVS of HBV origin, which correlated with increased sensitivities of (Abcam) were used as primary Abs. primary murine hepatocytes isolated from HBX knock-in trans- genic mice upon vesicular stomatitis virus (VSV) infections. Mice and cells HBX knock-in mice were generated as previously described (36). Primary murine hepatocytes from wild-type (WT) and HBX transgenic mice were Materials and Methods isolated and cultured as described previously (19). Cell culture and transfections Viral infections HEK293, HepG2, HepG2215, and HepG2-1117 cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS VSV, originally obtained from Dr. W. Chen (Institute of Pathology, Beijing, (HyClone Laboratories, Logan, UT). Cells were treated with MG132 China), was harvested from cell culture supernatants of BHK-21 cells, and (Sigma-Aldrich, St. Louis, MO) as noted in the text. HepG2-1117 cells virus titers were determined by plaque formation on Vero cells. Primary mu- contain 1.05-fold HBV genome (subtype ayw) under inducible Tet-off con- rine hepatocytes were infected with the equivalent of 10 ml of VSV stock in trol (33). Myc-tagged HBX and their mutants, Myc-tagged HBV X, S, S1, serum-free medium (1 ml/well) for 1 h at 37˚C. The infecting medium was and C, were expressed by cloning the genes into the pcDNA3-based vector then removed and replaced with 1 ml of normal growth medium. Cell super- (Invitrogen). HBV DNA genome head-to-tail dimer was provided by Prof. natants were recovered 24 h postinfection, and virus titers were determined Y. Wang in our laboratory. HBX-deleted HBV-2 DNA was generated by by plaque formation on Vero cells. HepG2-1117 cells were infected for 20 h two-step PCR using a QuikChange kit (Strategene, La Jolla, CA). GST with VSV (multiplicity of infection of 0.002) but were not killed; cells were fusion proteins were generated by expression in pGEX4T-2-based vectors fixed and then stained with amino black. (Amersham Biosciences, Piscataway, NJ) in Escherichia coli BL21 (DE3). Flag-MAVS plasmid was provided by Zhijian Chen (University of Texas Results Southwestern Medical Center, Dallas, TX), and NF-kB–Luc, IFN-b–Luc, Activation of IFN-b expression by cytosolic dsDNA is blocked or IRF-3–Luc reporter gene constructs were provided by Li Li (Biotechnology Institute of China, Beijing, China). MAVS mutants were by HBV cloned into pcDNA3 using overlap extension PCR. RecentreportssuggestthatHAVorHCVinfectionblockstheinduction Immunoprecipitation and immunoblot analysis of IFN-b synthesis by dislodging MAVS from the mitochondria. To determine whether RIG-I–MDA5 signaling was disrupted by HBV Immunoprecipitation and immunoblot analysis were performed as previ- ously described (34). Anti-MAVS (Abcam, Cambridge, MA), anti-Myc, infection, we ectopically expressed N–RIG-I or MAVS together with HRP-conjugated anti-Flag (Sigma-Aldrich), anti-GFP (Santa Cruz Biotech- a luciferase reporter driven by the IFN-b promoter (IFN-b–Luc) in nology, Santa Cruz, CA), or anti–b-tubulin (Sigma-Aldrich) Abs were used. HepG2 cells transfected with a head-to-tail dimer of HBV genome Subcellular fractionation (HBV-2 DNA). Previous studies have reported that HBV-2 DNA transfection supports the production of HBV particles in HepG2 cells. Cells were washed 36 h after transfection in hypotonic buffer (10 mM Tris- When using HepG2 cells, we also found that cells transfected with HCl [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, protease inhibitors) and then homogenized in the same buffer by bouncing 20 times. The homogenate was HBV-2 DNA continuously secreted HBsAg into the supernatant centrifuged at 500 3 g for 5 min to remove nuclei and unbroken cells. The (Supplemental Fig. 1), suggesting that the transfected HBV DNA supernatant was centrifuged again at 5000 3 g for 10 min to generate mem- could replicate and producevirus particles. We thus analyzed whether brane pellets containing mostly mitochondria and cytosolic supernatant. HBV could affect type I IFN signaling using the HBV DNA- Protein-binding assays transfected HepG2 cells. As shown in Fig. 1A,bothMAVSand N–RIG-I activated the IFN-b promoter in HepG2 cells transfected In GST pull-down experiments, cell lysates were incubated for 2 h at 4˚C with 5 mg purified GST or GST fusion proteins bound to glutathione beads. with empty vector, whereas these responses were substantially The absorbates were washed with lysis buffer and then subjected to SDS- reduced in HepG2 cells transfected with the HBV-2 DNA. These PAGE and immunoblot analysis. An aliquot of the total lysates (5%, v/v) results suggest that HBV DNA blocks RIG-I–MDA5 signaling. was included as a loading control on the SDS-PAGE. Poly(dAT:dAT) is a synthetic dsDNA that mimics dsDNA virus, Luciferase reporter assays which has previously been shown to induce IFN-b through RIG-I– MDA5 (11). In agreement with this observation, we also demon- HepG2 cells were transfected with 0.2 mg of the luciferase reporter pNF-kB– Luc, IFN-b–Luc, or IRF-3–Luc plus 0.02 mgoftheRenilla reporter pRL-TK, strated that IFN-b response to the transfected poly(dAT:dAT) or with or without various amounts of MAVS, RIG-I N-terminal CARD-like VSV infection was reduced by HBV DNA (Fig. 1A). Furthermore, domain mutant (N–RIG-I) expression vector, or poly(deoxyadenylate- we observed that HBV DNA suppressed the activation of NF-kBand thymidylate) [poly(dAT:dAT)]. Transfected cells were collected and luciferase IRF-3 reporters through MAVS, RIG-I, poly(dAT:dAT), and VSV activity was assessed as previous described (34). All experiments were re- infection (Fig. 1A). To better understand the interference of IFN-b peated at least three times. signaling by HBV, 105 HepG2 monolayer cells in a 6-cm plate were RNA analysis infected with HBV-positive serum containing 107 copies/ml HBVs or First-strand cDNA was generated from total RNA using random priming with control serum from uninfected individuals. Cells were washed and Moloney murine leukemia virus reverse transcriptase (Invitrogen). eight times to remove the excess of viral inputs 2 h postinfection 1160 HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY

FIGURE 1. HBV DNA blocks IFN-b induction by MAVS. A, HepG2 cells were transfected with plasmid-expressing HBV-2 DNA. After 36 h, cells were transfected with N–RIG-I, MAVS, or poly(dAT:dAT) together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. Cells transfected with HBV-2 DNA together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc were infected with VSV for 20 h. The Luc activity was measured 24 h later and normalized for transfection efficiency. B, HepG2 cells were infected with HBV-positive serum containing 107 copies/ml HBV. Cells were washed eight times to remove excess viral inputs 2 h postinfection and were then transfected with poly(dAT:dAT) together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. Serum from uninfected individuals was used as a control. The Luc activity was measured 24 h later and normalized for transfection efficiency. C, HepG2-1117 cells were infected with VSV with or without doxcycline. Protein extracts were then resolved by SDS-PAGE (upper panel) or native gel electrophoresis (lower panel). Phosphorylation or di- merization of IRF-3 was detected by IB with an IRF-3 Ab. D, The experiments were carried out as in B, except that HBV X, S, S1, and C plasmid was transfected together with poly(dAT:dAT). Cell lysates were immunoblotted with anti-Flag Ab. The Luc activity was measured 24 h later and normalized for transfection efficiency. E, HepG2 cells were transfected with HBV–2-DNA or HBV-2 DNA-4HBX. After 36 h, cells were transfected with poly (dAT:dAT) together with IFN-b–Luc. The Luc activity was measured 24 h later and normalized for transfection efficiency. Cell lysates were immunoblotted with anti-HBX Ab. F, HepG2 cells were transected with plasmids expressing HBX together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc and were then were infected with VSV. The Luc activity was measured 24 h later and normalized for transfection efficiency. G, HepG2 cells were transfected with the HBV-2 DNA or HBX expression plasmid together with the expression vectors for RIG-I, DAI, MAVS, TBK1, or IKKε. Cell lysates were immunoblotted with anti-Flag or anti-Myc Ab. b-Tubulin was used as an equal loading control. For A, B,andD–F, the Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. IB, immunoblotting. The Journal of Immunology 1161 and were then transfected with poly(dAT:dAT) together with IFN-b– levels induced by VSV were lower in HepG2-1117 cells without luc, NF-kB–Luc, or IRF-3–Luc. We found that HBV also inhibited doxcycline. In agreement with these data, immunoblotting experi- the induction of IFN-b, IRF-3, and NF-kB by cytosolic poly(dAT: ments also showed that VSV-activated IRF-3 phosphorylation was dAT) (Fig. 1B). HepG2-1117 cells contain Tet-off HBV-1.05 ge- inhibited by HBVand in HepG2-1117 cells without doxcycline (Sup- nome. Supplemental Fig. 1C shows that IFN-b, IRF-3, and NF-kB plemental Fig. 1B,1C). To delineate the mechanism underlying the

FIGURE 2. HBX associates with MAVS. A, HEK293 cells were transfected with Flag-MAVS–expressing plasmid. The GST-fusion protein absorbates from cell lysates were analyzed by immunoblotting with anti-Flag Ab (top panel). Loading of the GST proteins was assessed by Coomassie blue staining (bottom panel). B, HEK293 cells were cotransfected with Flag-MAVS and Myc-HBX expression plasmid or Flag-vector, and anti-Flag or IgG immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab. C, HEK293 cells were transfected with Flag-HBX expression plasmid or Flag-vector, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-MAVS or anti-Flag Ab. Lysates from HEK293 cells transfected with HBV-2 DNAwere subjected to immunoprecipitation with anti-MAVS or IgG, fractionated by SDS-PAGE, and subsequently analyzed by immunoblotting with anti-MAVS Ab or anti-HBX Ab. D, HEK293 cells were cotransfected with Myc-HBX expression plasmid and Flag-MAVS or with Flag-MAVS mutants, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab. E, HEK293 cells were cotransfected with Flag-MAVS expression plasmid and Myc-HBX or with Myc-HBX mutants, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab. F, HEK293 cells were transfected with Myc-HBX (73–154) or Myc-HBX (73–154 C115A) plasmid, and mitochondria and nuclear and cytosolic fractions were analyzed by immunoblotting with anti- Myc, cytochrome c, or lamin B1 Ab. G, HEK293 cells were cotransfected with Flag-MAVS expression plasmid and Myc-HBX (73–154) or with Myc-HBX (73– 154 C115A) plasmid, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab. 1162 HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY reduced IFN-b signaling, expression constructs for four HBV- TBK1 proteins from HEK293 cells transfected with either empty encoded proteins (X, S, S1, and C) were generated, and one of these vector, HBV-2 DNA, or HBX expression plasmid. As shown in four constructs was used to cotransfect HepG2 cells together with Fig. 1G, MAVS protein abundance was substantially reduced by poly(dAT:dAT). As shown in Fig. 1D, the induction of IFN-b by HBV-2 DNA or HBX, suggesting that MAVS is a target of HBV poly(dAT:dAT) was only inhibited by HBX. To confirm the and that HBX is a potential factor that may interfere with the inhibitory role of HBX in IFN-b signaling, we deleted the HBX stability of MAVS and thus IFN-b signaling. gene in HBV-2 DNA. We found substantially higher levels of IFN-b induction in the cells thus transfected compared with those HBX interacts with MAVS transfected with HBV-2 DNA (Fig. 1E). HBX introduction also Our observation that HBX could downregulate MAVS, as well as blocked IFN-b promoter activation by VSV induction (Fig. 1F), the fact that both MAVS and some portion of HBX reside in the suggesting that HBX was able to independently inhibit IFN-b mitochondria compartment, raised the possibility that HBX might activation. physically interact with MAVS. To test this possibility, lysates from To determine the modulation of protein abundance of the RIG-I– HEK293 cells were incubated with GSTor GST-HBX fusion protein. MAVS pathway by HBV DNA and HBX, immunoblotting was We found that MAVS bound to GST-HBX but not to GST (Fig. 2A), used to analyze the levels of RIG-I, MAVS, DAI, IKKε, and demonstrating an in vitro interaction between HBX and MAVS.

FIGURE 3. HBX blocks IFN-b induction by MAVS. A, HEK293 cells were transfected with increasing amounts of HBX expression vector. After 36 h, cells were transfected with MAVS together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. The Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. B, The experiments were carried out as in A, except that N–RIG-I plasmid was transfected in lieu of MAVS. C, HEK293 cells were transfected with plasmid-expressing HBX. After 36 h, cells were transfected with poly(dAT:dAT). RNA was extracted and IFN-b mRNA was analyzed by quantitative RT-PCR. D, The experiments were carried out as in A, except that IKKε plasmid was transfected in lieu of MAVS. The Journal of Immunology 1163

FIGURE 4. HBX downregulates MAVS. A, HEK293 cells were transfected with plasmids expressing increasing amount of Myc-HBX (0.25, 0.5, and 0.75 mg). Whole-cell lysates were analyzed by immunoblotting with anti-MAVS or anti-Myc Ab. b-Tubulin was used as an equal loading control. B,Myc-HBX or Myc-HBX 73–154 C115A plasmid was transfected with the expression vector encoding HA-tagged ubiquitin. Cells were grown in DMEM containing MG132 (20 mM) for 6 h. Anti-MAVS immunoprecipitates were analyzed by immunoblotting with anti-HA Ab, whole-cell lysates were subjected to immunoblotting with anti-Myc and anti-MAVS Ab, and b-tubulin was used as an equal loading control. C, Myc-HBX or empty vectors were cotransfected with plasmids encoding Flag-MAVS or its mutants. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-Myc Ab. Ten nanograms of plasmid encoding GFP was transfected as a loading control, and whole-cell lysates were analyzed by immunoblotting with anti-GFP Ab. D, Myc-HBX or empty vectors were cotransfected with plasmids encoding Flag-MAVS or its mutants together with IFN-b–Luc. The Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. E, Myc-HBX was transfected with the expression vector encoding Flag-MAVS or Flag-MAVS K136R mutant together with HA-tagged ubiquitin. Cells were grown in DMEM containing MG132 (20 mM) for 6 h. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-HA Ab, whole-cell lysates were subjected to immunoblotting with anti-Myc and anti-Flag Ab, and b-tubulin was used as an equal loading control. F, HEK293 cells were cotransfected with plasmids expressing Flag-MAVS and Myc-HBX or its mutants. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-Myc Ab, and b-tubulin was used as an equal loading control. G, HEK293 cells were cotransfected with Flag-MAVS, Myc-HBX, or HBX mutants together with IFN-b–Luc. The Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. 1164 HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY

To test if HBX binds to MAVS in mammalian cells, HEK293 cells endogenous MAVS protein level. Increasing amounts of the HBX were transfected with Myc-tagged HBX and Flag-tagged MAVS. expression vector were transfected into HEK293 cells. A striking Immunoblotting analysis of anti-Flag immunoprecipitates with anti- reduction in the abundance of endogenous MAVS with overex- Myc Ab showed a significant association between Flag-MAVS and pressed HBX was found, and this reduction of MAVS by HBX was Myc-HBX (Fig. 2B). To further characterize the endogenous MAVS also dose-dependent (Fig. 4A). As a control, increasing amounts of interaction with HBX, lysates from HEK293 cells transfected with HBV S Ag expression vector did not change the level of endog- Flag-HBX were subjected to immunoprecipitation with anti-Flag enous MAVS (data not shown), indicating that this effect is spe- Ab by immunoblotting with an anti-MAVS Ab, and MAVS was cific to HBX. Similarly, the HepG2-1117 cell line, which contains found in Flag-HBX–transfected immunoprecipitates but not in the an inducible Tet-off HBV-1.05 genome, showed reduced endoge- nontransfected control immunoprecipitations (Fig. 2C). Addition- nous MAVS levels without doxcyline. Quantitative RT-PCR ally, lysates from HEK293 cells transfected with HBV-2 DNA were revealed that HBX overexpression did not change MAVS mRNA subjected to immunoprecipitation. HBX was shown to interact with levels (data not shown), suggesting that HBX downregulates MAVS endogenous MAVS in the HBV-2 DNA-transfected cells (Fig. 2C), by posttranscriptional modification. Indeed, the estimated half-life suggesting that HBX interacts with MAVS in vivo. of the MAVS was significantly longer than that of MAVS in the To map the domains of MAVS responsible for the interaction with presence of HBX (data not shown). To further delineate the mech- HBX, Flag-tagged proteins containing a deletion of various regions anisms responsible for the HBX-mediated MAVS degradation, of MAVS (CARD, pro, and transmembrane [TM] domain) were HEK293 cells were cotransfected with plasmids expressing Myc- prepared, and the ability of each of these mutants to interact with HBX or Myc-HBX 73–154 C115A together with HA-ubiquitin in HBX was analyzed by immunoprecipitation. Fig. 2D shows that the presence of proteasome inhibitors MG132 (20 mM for 6 h), and Myc-tagged HBX interacted with full-length and pro mutant MAVS MAVS was then immunoprecipitated by anti-MAVS Ab and blotted but not with the TM mutant MAVS. The CARD MAVS mutant also with anti-HA Ab. As shown in Fig. 4B, WT HBX introduction led interacted with HBX with lower affinity. Thus, the interaction with to an increased steady level of MAVS ubiquitination whereas HBX HBX is specific for the CARD and TM domains of MAVS. 73–154 C115A abolished this effect. These results indicate that To define the interacting regions of HBX on MAVS, Myc-tagged HBX may have a major role in MAVS ubiquition and its protea- proteins containing N-terminal and C-terminal HBX were prepared, some-mediated degradation. and the ability of each of these proteins to interact with MAVS was To further dissect the possible HBX-mediated ubiquitination sites analyzed by immunoprecipitation. The C-terminal 73–154 residues inMAVS,14mutants weregenerated by substituting the 14 lysineres- of HBX did not affect its ability to interact with MAVS, whereas idues on the MAVS molecule individually with arginine (KRR) and the N-terminal HBX 1–72 domain totally eliminated its ability to then tested for their stability by introducing them with HBX into bind MAVS (Fig. 2E). Thus, the interaction with MAVS requires HEK293 cells. As shown in Fig. 4C, HBX had little effect on the the C-terminal domain of HBX. A previous report revealed the key cellular abundance of MAVS K136R mutations and modestly de- amino acid for mitochondrial targeting was mapped to be HBX C creased the cellular abundance of MAVS K297R and K420R muta- terminus 111–116 aas (37); when Cys115 of HBX was mutated to tion. In contrast, HBX led to a sharp decrease on other MAVS KRR alanine (HBX C115A), the mitochondrial targeting property of mutations and WT MAVS as well. Furthermore, MAVS K136R- HBX was abrogated. Consistent with this report, we found that induced IFN-b activation was only partially inhibited by HBX. The when Cys115 of 73–154 C-terminal HBX was substituted with ala- extent of HBX suppression on MAVS K297R and K420R mutation- nine (HBX 73–154 C115A), its mitochondria localization was par- induced IFN-b activation was much lower than that achieved with tially abrogated and its interaction with MAVS was much weaker WT MAVS or other MAVS KRR mutations (Fig. 4D). In agreement than for the intact HBX C-terminal (HBX 73–154) (Fig. 2F,2G). with this observation, HBX induced increased ubiquitin levels of WT MAVS with less effect on the MAVS K136R mutant (Fig. 4E). b HBX blocks MAVS-mediated IFN- induction These results suggest that Lys136 is one of the critical sites for To better understand how HBX blocks IFN-b signaling through HBX-mediated ubiquitination in MAVS proteins. MAVS, increasing amounts of HBX expression vector and 0.5 mg of MAVS expression construct were transfected into HEK293 cells together with IFN-b–Luc. As was shown in Fig. 3A, as low as 0.5 mg of HBX expression construct was sufficient to exert a potent repression of IFN-b response, and the extent of repression increased with increasing amounts of HBX expression, suggesting that HBX inhibited the induction of IFN-b by MAVS in a dose- dependent manner. We also observed similar repression of MAVS- induced activation of NF-kB and IRF-3 reporters by HBX (Fig. 3A). We then transfected cells with poly(dAT:dAT) or with an expression vector encoding N–RIG-I together with HBX. Fig. 3B shows that the induction of IFN-b by RIG-I or poly(dAT:dAT) (data not shown) was also inhibited by HBX. As expected, IFN-b mRNA levels were reduced sharply in cells containing the HBX expression plasmid (Fig. 3C). In contrast, the induction of IFN-b by IKKε was not affected (Fig. 3D). Collectively, these data suggest that HBX FIGURE 5. HBX affects the kinetics of RIG-I–MAVSsignaling. HEK293 cells were cotransfected with Myc-MAVS and Flag–RIG-I expression plas- inhibits RIG-I–MAVS signaling at the level of MAVS or at a step ε mids together with the expression vector encoding GFP-HBX or empty downstream of MAVS, but upstream of IKK . vector. After 24 h, cells were treated with VSV for indicated times. Cells HBX downregulates MAVS were then harvested at different times after VSV infection as indicated. Anti- Flag or IgG immunoprecipitates were analyzed by immunoblotting with anti- To further elucidate the mechanism for the inhibitory effect of Myc or anti-Flag Ab. HBX expression was monitored by immunoblotting HBX on antiviral signaling, we examined the effect of HBX on the using GFP Ab. b-Tubulin was used as an equal loading control. The Journal of Immunology 1165

We also examined whether HBX 73–154 C115A could also for MAVS downregulation and MAVS-mediated antiviral signaling affect the protein stability of MAVS. In contrast to the HBX inhibition. 73–154 C-terminal domain, the cellular abundance of MAVS in 73–154 C115A-expressing cells was unchanged (Fig. 4F). Addi- HBX affects the kinetics of RIG-I–MAVS signaling tionally, the extent of MAVS-induced IFN-b repression by HBX Our studies have shown that HBX inhibits RIG-I–MAVS signaling 73–154 C115A was lower than that achieved with WT HBX or by downregulating MAVS, indicating that HBX might affect the with the HBX 73–154 C-terminal domain (Fig. 4G). These results kinetics of the MAVS–RIG-I association upon virus infection. indicate that interaction between HBX and MAVS is responsible HEK293 cells were cotransfected with Myc-MAVS and Flag–RIG-I

FIGURE 6. Expressions of MAVS proteins in liver cancer patients and their association with HBV disease. A, HepG2, HepG2215, and primary murine hepatocytes isolated from HBX transgenic mice or control mice were cultured, and whole-cell lysates were analyzed by immunoblotting with anti-MAVS. b-Tubulin was used as an equal loading control. HepG2-1117 cells containing 1.05-fold HBV genome under Tet-off control were cultured with or without doxcycline for 7 d, whole-cell lysates were analyzed by immunoblotting with anti-MAVS, and b-tubulin was used as an equal loading control. HepG2-1117 cells were cultured with or without doxcycline for 7 d and transfected with Flag-MAVS together with HA-Ub plasmid, anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-HA Ab, whole-cell lysates were subjected to immunoblotting with anti-Flag Ab, and b-tubulin was used as an equal loading control. B, Representative immunoblot of MAVS protein in HBV-induced HCC (C1–C4), HBV-irrelevant HCC (C5–C8), and in their matched controls (N1–N4). Immu- noblot of HBX expression was also analyzed in C1–C8 samples. C, Representative immunohistochemical staining of MAVS expression in normal donor livers (1), HBV-irrelevant cirrhosis (2), HBV-irrelevant HCC (3), HBV-induced cirrhosis (4), and in HBV-induced HCC (5). 1–5, Original magnification 3200. 1166 HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY expression plasmids together with the expression vector encoding HCC, 86% expressed lower levels of MAVS (13/15), suggesting GFP-HBX or empty vector. After 24 h, cells were treated with VSV a negative correlation between HBX expression and MAVS pro- and then harvested at different times as indicated. As shown in tein level. Fig. 5, the interaction of MAVS and RIG-I was enhanced by VSV MAVS protein levels were also measured by immunohisto- infection, whereas the introduction of HBX not only eliminated chemistry in normal donor livers (n = 14), in livers of HBV- this enhancement but it also reduced MAVS–RIG-I interaction. Be- unrelated cirrhosis and HCC (n = 30), in livers with cirrhosis cause this RIG-I–MAVSinteraction is required for RIG-I–mediated caused by HBV infection (n = 22), and in HCC caused by HBV IFN-b signaling, the disruption of this interaction by HBX would infections (n = 24). Results showed that 78.6% (11/14) of healthy squelch downstream signaling. Collectively, these observations livers, 76.6% (23/30) of HBV-unrelated liver diseases, and 30.4% solidify the negative regulatory role of HBX in MAVS-mediated (14/46) of HBV-related liver diseases stained strongly positive for antiviral responses, and they demonstrate that HBX exerts its in- MAVS proteins (Fig. 6C). The overall differences in MAVS- hibition of virus induced RIG-I–MAVS signaling through downre- positive staining between HBV-induced liver disease groups and gulation of MAVS. the other three HBV-irrelevant groups including normal controls were significant (30.4 versus 77.6%). Taken together, these data Expression of MAVS proteins in liver cancer patients and their suggest a negative correlation between MAVS protein and HBV association with HBV disease liver disease. To examine the status of MAVS in the presence of HBV, we performed Western blotting analysis to examine the expression of HBX regulates RIG-I-dependent antiviral cellular responses MAVS protein levels in HepG2-derived, HBV DNA-transfected We next sought to determine whether HBX regulates replication of HepG2215 cells, HepG2-1117 cells that contain Tet-off HBV- VSV virus replication, as RIG-I–mediated IFN-b signaling is 1.05 genome, and in primary murine hepatocytes isolated from critical in restricting replication of these RNA viruses. We HBX transgenic mice. It is evident that MAVS was present at therefore used VSV to infect HepG2-1117 cells with or without low levels in HepG2215 cells and in HepG2-1117 cells without doxcycline. The results showed that HepG2-1117 without doxcy- doxcycline; additionally, transfected MAVS in HepG2-1117 cells cline increased the production of VSV ~50% (Fig. 7A). Further- showed increased ubiquitin modification when HBX expression more, primary murine hepatocytes isolated from HBX knock-in was induced without doxcycline (Fig. 6A). Consistent with this mice or the WT littermate control mice were cultured and infected finding, primary hepatocytes isolated form HBX knock-in trans- with VSV virus. Fig. 7B shows that exogenous expression of HBX genic mice also showed reduced endogenous MAVS level com- increased the production of infectious VSV. Taken together, these pared with WT littermate control mice (Fig. 6A). results provide more evidence that HBX modulates the innate We next examined the expression of MAVS proteins in HBV- antiviral cellular response by acting as a negative regulator of induced HCC (n = 20) and matched healthy controls (n = 20), as RIG-I–mediated IFN-b signaling infection. well as in HBV-unrelated HCC (n = 20) and matched healthy controls (n = 20). Our results showed that 80% (16/20) of HBV- Discussion induced HCC expressed lower levels of MAVS (Fig. 6B) and that The targeting of MAVS by both the cysteine protease of HAV and 10% (2/20) of HBV-unrelated HCC expressed lower levels of the serine protease of HCV represents a remarkable example of MAVS (Fig. 6B). The occurrence of reduced MAVS expression convergent virus evolution (38) and provides strong evidence for between the HBV-induced HCC and the HBV-unrelated HCC was the importance of MAVS to host control of virus infections in the significant. To further delineate the correlation between HBX and liver. In this study, we show that both HBV DNA or HBV suppressed MAVS, HBX protein expression was also analyzed in those 20 poly(dAT:dAT)-activated IFN-b production in hepatocytes. Addi- HBV-induced HCC samples. Seventy-five percent (15/20) showed tionally, MAVS interacts with HBX and is indeed the target of HBX positive HBX expression (Fig. 6B); among the 15 HBX-positive protein. Specifically, we found that HBX promoted the degradation

FIGURE 7. HBX regulates RIG-I–dependent antiviral cellular responses. A, HepG2-1117 cells with or without doxcycline were infected for 20 h with VSV (multiplicity of infection of 0.002) but not killed; cells were fixed and then stained with amino black. B, Primary murine hepatocytes were infected with the equivalent of 10 ml of VSV stock in serum-free medium (1 ml/well) for 1 h at 37˚C. The infecting medium was then removed and replaced with 1 ml of normal growth medium. Cell supernatants were recovered 24 h postinfection, and virus titers were determined by plaque formation. The Journal of Immunology 1167 of MAVS through Lys136 ubiquitin in MAVS protein, thus prevent- characterized by the production of type I IFN-a/b cytokines and the ing the induction of IFN-b. We also show that the enhanced activation of NK cells. HBV replication can be efficiently limited interaction of MAVS and RIG-I by VSV infection was eliminated by a and b IFN, but data on acutely infected chimpanzees suggest in the presence of HBX. Meanwhile, MAVS protein abundance was that such antiviral cytokines are not triggered by HBV replication reduced in patients with liver cancer that was caused by HBV in- (44). A further characteristic of HBV in relationship to early host fection, and primary hepatocytes isolated from HBX transgenic defense mechanisms resides in the lack of IFN-a and IFN-b pro- mice showed hypersensitivity to VSV infection. These studies in- duction. HBV might have evolved strategies to escape the initial dicate that HBV can target RIG-I–MAVS signaling by HBX- antiviral defense mechanisms activated by the TLR system or RIG- mediated MAVS downregulation and thereby attenuate the antiviral I–MDA5 pathway. Previous reports have shown that HBV almost response of the innate immune system. completely abrogated TLR-induced antiviral activity (20). Cheng HBX, a virally encoded protein of 154 aas, has been shown to be et al. (45) demonstrated that recombinant HBsAg inhibits LPS/ essential for the establishment of HBV infection in vivo. Its gene TLR4-induced NF-kB activation, leading to reduced COX-2, IL- product also activates a variety of viral and cellular promoters in 18, and IFN-g production in the human monocytic cell line THP-1. diverse cell types. Although HBX does not bind to dsDNA, it does Other studies have shown lower TNF-a levels in HBeAg-positive regulate transcription of a variety of cellular and viral genes by patients compared with HBeAg-negative patients (46, 47). In this interacting with cellular proteins and/or components of signal study, we analyzed the involvement of MAVS in HBV infection by transduction pathways (39, 40). The interaction of HBX with these using liver samples from HBV-induced cirrhosis and from HCC proteins leads to activation of signal transduction pathways in- patients. We demonstrated that MAVS was downregulated mark- cluding Ras/Raf/MAPK, protein kinase C, and Jak1-STAT. HBX edly in livers from these patients, with the decrease in MAVS has been reported to activate TNF-mediated NF-kB signaling by having been correlated with HBX expression in HCC liver tumors. inducing the degradation of IkB-a. We show herein that HBX Several studies have demonstrated the pivotal position of RIG-I– suppressed intracellular IFN-b signaling through MAVS degra- MAVS signaling in host responses against a number of viruses (48, dation. Thus, it is very likely that HBX acts as a regulator of 49). Recent research has indicated that the induction of TLR- and the antiviral pathway through different mechanisms under differ- RIG-I–MDA5-mediated host cellular innate immune responses by ent stimuli. overexpression of the three pattern recognition receptors adaptors, MAVS contains an N-terminal CARD-like domain and IPS-1, TRIF, and MyD88, dramatically reduces the levels of HBV a C-terminal transmembrane domain that target the protein to the mRNA and DNA in both HepG2 and Huh7 cells. These observa- outer mitochondrial membrane. The mitochondrial-targeting trans- tions provide more evidence that HBV might evolve multiple stag- membrane domain is essential for MAVS signaling, thus implicat- iest to evade TLR-dependent and -independent signaling pathways; ing a new role for mitochondria in innate immunity. Previous further understanding of the nature of these mechanisms should reports have shown that HBX regulates fundamental aspects of yield novel strategies for developing antivirals that evoke responses mitochondrial physiology, including downregulating mitochondrial to eliminate HBV infection. and increasing the cellular abundance of reactive oxygen species. We have shown herein that HBX regulates the stability of Acknowledgments a mitochondrial protein that is essential for innate immunity, thus We thank Zhijian Chen (University of Texas Southwestern Medical Center, shedding new light on the physiological significance of HBX, which Dallas, TX) for providing the MAVS plasmid. may contribute to liver disease associated with HBV-persistent infection. Previous reports have also shown that MAVS proteins Disclosures undergo proteasomal degradation by ubiquitin E3 ligase RNF125- The authors have no financial conflicts of interest. mediated ubiquitin conjugation (41). 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