Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex - PubMed (original) (raw)

Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex

Xianmiao Wang et al. Cell Mol Immunol. 2010 Sep.

Abstract

Viral RNAs produced during viral infection are recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). A central adapter protein downstream of RIG-I and MDA5 is the mitochondrial membrane protein virus-induced signaling adaptor (VISA), which mediates the induction of type I interferons (IFNs) through the activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) and IFN-regulatory factor-3 (IRF3). Here we found that hepatitis B virus (HBV)-encoded X protein (HBx) acts as an inhibitor of virus-triggered IRF3 activation and IFN-beta induction. Reporter and plaque assays indicate that HBx inhibits signaling by components upstream but not downstream of VISA. Immunoprecipitation experiments indicate that HBx interacts with VISA and disrupts the association of VISA with its upstream and downstream components. These findings suggest that HBx acts as a suppressor of virus-triggered induction of type I IFNs, which explains the observation that HBV causes transient and chronic infection in hepatocytes but fails to activate the pattern recognition receptor-mediated IFN induction pathways.

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Figures

Figure 1

HBx inhibits virus-triggered IRF3 activation and IFN-β induction. (a) HBx inhibits SeV-induced activation of the IFN-β promoter in a dose-dependent manner. The 293 cells (1×105) were transfected with the IFN-β promoter reporter plasmid (0.1 µg) and the indicated amount of expression plasmid for HBx. Twenty-four hours after transfection, cells were left uninfected or infected with SeV for 12 h before luciferase assays were performed. (b) Effects of HBV and mutant replicons on SeV-triggered activation of the IFN-β promoter. Experiments were performed as in (a). (c) HBx inhibits transcription of IFNB1 and RANTES genes. The 293 cells (1×105) were transfected with the indicated plasmids for 24 h. RT-PCR was performed 10 h after SeV infection. The quantitative software Quantity One was used to examine the amount of DNA in agarose gels; the IFN-B1, Rantes and ISG15 values were then normalized to GAPDH values. The specific values are used for graphic display. (d) HBx inhibits SeV-induced ISRE activation. The experiments were performed as in (a). (e) HBx inhibits SeV-induced dimerization of endogenous IRF3. The 293 cells (2×105) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were infected with SeV or left uninfected for 6 h. Cell lysates were separated by native (upper panel) or SDS (bottom two panels) PAGE and analyzed with the indicated antibodies. (f) HBx does not inhibit SeV-induced NF-κB activation. The experiments were performed as in (a). (g) HBx inhibits poly(I:C)-induced activation of the IFN-β promoter. The 293 cells (1×105) were transfected with the IFN-β reporter and HBx expression plasmids. Twenty-four hours after transfection, cells were transfected with poly(I:C) (0.5 µg/well) or left untransfected for 12 h before luciferase assays were performed. payw: a plasmid carrying a greater-than-length (129%) HBV genome (subtype ayw); payw*7: an HBx-deficient version of the same plasmid of payw. HBV, hepatitis B virus; HBx, HBV-encoded X protein; NF-κB, nuclear factor-kappaB; IFN, interferon; ISRE, IFN-stimulated response element; IRF, IFN-regulatory factor; PAGE, polyacrylamide gel electrophoresis; SeV, Sendai virus.

Figure 2

HBx inhibits virus-triggered signaling at the level of VISA. HBx inhibits activation of the IFN-β promoter mediated by RIG-I-N (a), MDA5-N (b), VISA (c) and MITA (d), but not TBK1 (e). The 293 cells (1×105) were transfected with the IFN-β promoter plasmid (0.1 µg) and the indicated expression plasmids. Luciferase assays were performed 24 h after transfection. (f) HBx inhibits virus-triggered ISRE activation at a level upstream of TBK1. Experiments were performed as in (a)–(e), with the exception that an ISRE reporter was used. (g) The role of HBx in the cellular antiviral response. The 293 cells (1×105) were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cells were infected with VSV (MOI=0.1) and supernatants were harvested at 12 hpi. Supernatants were analyzed for VSV production by standard plaque assay. HBx, HBV-encoded X protein; hpi, hours postinfection; IFN, interferon; ISRE, IFN-stimulated response element; MOI, multiplicity of infection; TBK1, TANK-binding kinase 1; VSV, vesicular stomatitis virus.

Figure 3

Analysis of the interactions between HBx and MDA5, VISA or TBK1. (a) HBx interacts with MDA5 and VISA but not TBK1. The 293 cells (2×106) were transfected with the indicated plasmids (8 µg each). Twenty-four hours after transfection, cells were lysed and coimmunoprecipitation experiments were performed. (b) HBx binds to the N-terminal CARD domain of MDA5. The experiments were performed as described in (a). (c) HBx interacts with the C-terminal domain (aa 360–540) of VISA. The experiments were performed as in (a). (d) Colocalization of HBx with MDA5 and VISA. The 293 cells were transfected with the indicated plasmids. Transfected cells were stained with MitoTracker Red and observed by confocal microscopy. aa, amino acid; HBx, HBV-encoded X protein; TBK1, TANK-binding kinase 1.

Figure 4

HBx disrupts the VISA-associated complex. Overexpression of HBx disrupts the interaction between VISA and MDA5 (a), RIG-I (b), MITA (c) and TBK1 (d). The 293 cells (2×106) were transfected with the indicated plasmids. Coimmunoprecipitation and immunoblot analysis were performed as in Figure 3a. In (a)–(d), while the expression levels of VISA and VISA-associated proteins were similar, the amount of coimmunoprecipitated VISA was much less after overexpression of HBx. HBx, HBV-encoded X protein; TBK1, TANK-binding kinase 1.

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Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex - PubMed (original) (raw)