GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS - PubMed (original) (raw)

GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS

Zihong Chen et al. J Virol. 2007 Jan.

Abstract

Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.

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Figures

FIG. 1.

GBV-B NS3/4A protease, but not YFV NS2B/3 or BVDV NS3/4A, inhibits SenV-induced activation of the IFN-β promoter. A. HEK293 cells were cotransfected with pIFN-β-Luc and pCMVβgal plasmids and plasmids encoding HCV NS3/4A, GBV-B NS3/4A, YFV NS2B/3, or BVDV NS3/4A, or a control vector. Twenty-four hours later, cells were either mock infected (empty bars) or infected with SenV at 100 HAU/ml for 16 h (solid bars) prior to lysis for both luciferase and β-galactosidase assays. Bars show relative luciferase activity normalized to β-galactosidase activity, i.e., IFN-β promoter activity. B. IFN-β promoter activity in tamarin hepatocytes (TH1-5s) transfected with the indicated amounts (in micrograms) of GBV-B NS3/4A-expressing plasmid supplemented with a control vector, to keep the total amount of DNA transfected constant, and then mock infected or infected with SenV for 16 h. C. Immunoblot analysis of TH1-14s cells transiently transfected with plasmids encoding WT GBV-B NS3/4A or an active site mutant, S139A, and then mock infected or infected with SenV. Note that cells transfected with the S139A mutant plasmid expressed unprocessed NS3-4A precursor (arrow). D. IFN-β promoter activity in tamarin hepatocytes (TH1-14s) transfected with plasmid DNAs expressing GBV-B NS3/4A, the S139A mutant GBV-B NS3/4A, or with a control vector and mock infected or infected with SenV for 16 h.

FIG. 2.

GBV-B NS3/4A protease disrupts virus or constitutive active RIG-I CARD (N-RIG)-induced activation of both IRF-3 and NF-κB. A. TH1-14s cells were cotransfected with plasmids encoding luciferase under the control of either an IRF-3-dependent PRDIII-I promoter (four-time repeat of the PRDIII-I element, left) or an NF-κB-dependent PRDII promoter (right), pCMVβgal, and plasmids encoding HCV NS3/4A, GBV-B NS3/4A, or GBV-B NS3/4A S139A or control vector. Twenty-four hours later, cells were either mock infected or infected with SenV at 100 HAU/ml for 16 h prior to lysis for both luciferase and β-galactosidase assays. B. Constitutively active RIG-I CARD (N-RIG)-induced PRDIII-I (left) and PRDII (right) promoter activities in HEC1B cells cotransfected similarly to those represented in panel A. In these experiments, cotransfection of an N-RIG-encoding plasmid or empty vector was substituted for SenV infection or mock infection, respectively, and cells were harvested at 24 h posttransfection.

FIG. 3.

GBV-B NS3/4A disrupts the endogenous IFN response to virus infection in HeLa GBpro-10 cells with tetracycline-regulated, conditional expression of GBV-B NS3/4A. A. Confocal microscopy of IRF-3 subcellular localization in HeLa GBpro-10 cells repressed (+tet) or induced (-tet) for GBV-B NS3/4A expression and mock infected (left) or infected with SenV (right) for 16 h. Nuclei were counterstained with 4′,6′-diamidino-2- phenylindole. B. HeLa GBpro-10 cells were cultured to repress or induce GBV-B NS3/4A expression for 3 days, followed by mock infection or infection with 100 HAU/ml of SenV for 16 h prior to immunoblot analysis of whole-cell extracts for IRF-3, ISG56, SenV, or GBV-B NS3. The arrow in the IRF-3 panel indicates the hyperphosphorylated form of IRF-3 (IRF3-P). A nonspecific band (*) detected by anti-ISG56 antiserum indicates equal loading. C. Real-time RT-PCR analysis of IFN-β (left) and IL-6 (right) mRNA transcripts in HeLa GBpro-10 cells repressed or induced for GBV-B NS3/4A expression and mock infected or infected with SenV. mRNA abundance was normalized to cellular 18S rRNA.

FIG. 4.

GBV-B NS3/4A protease targets MAVS/IPS-1/Cardif/VISA for proteolysis. A. Immunoblot analysis of endogenous MAVS in UNS3-4A-24 cells (left) and HeLa GBpro-10 cells (right) with (-tet) or without (+tet) induction of HCV and GBV-B NS3/4A, respectively. Open circles indicate intact MAVS, while solid circles indicate cleaved MAVS. B. TH1-14s cells were mock transfected (lane 4), transfected with a plasmid encoding MAVS (lane 1), or cotransfected with plasmids encoding MAVS and either GBV-B NS3/4A (lane 2) or S139A mutant GBV-B NS3/4A (lane 3) for 24 h prior to harvest for immunoblot analysis of whole-cell extracts with MAVS antiserum (upper panel) or GBV-B NS3 antibodies (lower panel). C. HeLa GBpro-10 cells induced or repressed for GBV-B NS3/4A expression were transfected with WT or the indicated mutants of N-terminally Flag-tagged MAVS. Forty-eight hours later, MAVS protein was detected with anti-Flag antibody by immunoblot analysis of whole-cell extracts. D. Activation of the IFN-β promoter by ectopic expression of IKKɛ, IRF-3 5D, or WT or C508R mutant MAVS in HEK293 cells in the absence (empty bars) or presence (solid bars) of ectopic coexpression of GBV-B NS3/4A.

FIG. 5.

Cleavage of MAVS/IPS-1/Cardif/VISA by GBV-B NS3/4A causes its redistribution from mitochondria to the cytosol. A. HeLa GBpro-10 cells induced or repressed for GBV-B NS3/4A expression were transfected with a vector encoding N-terminally GFP-tagged MAVS (GFP-MAVS) and analyzed by confocal microscopy 24 h later. Mitochondria were stained with Mitotracker Red. B. Immunoblot analysis of GFP-MAVS (using GFP antibody) and GBV-B NS3 in whole-cell extracts of HeLa GBpro-10 cells treated as for panel A. Open circles indicate intact GFP-MAVS, while solid circles indicate cleaved GFP-MAVS. C. Immunoblot analysis of MAVS (using anti-MAVS antiserum), a mitochondrial inner membrane protein, complex I 39-kDa subunit (CI39), and GBV-B NS3 in crude mitochondrial fractions isolated from HeLa GBpro-10 cells induced (-tet) or repressed (+tet) for GBV-B NS3/4A expression.

FIG. 6.

The virus-induced IFN response is abrogated in cells supporting the autonomous replication of GBV-B subgenomic RNAs due to MAVS cleavage. A. MAVS is cleaved in cells containing GBV-B replicons. In IFN-cured cells (− replicon lanes), an intact MAVS-immunoreactive product (arrow with open circle) was found in whole-cell extracts prepared in RIPA buffer, as well as in membranes collected after mild lysis using reporter lysis buffer. In cells containing the GBV-B replicon RNA (RepGBNeo5m#15), a protein with a slightly lower molecular mass (arrow with solid circle) was found in RIPA-lysed extracts, as well as in the supernatant from mildly lysed cells, consistent with NS3/4A cleavage and redistribution of MAVS from mitochondria to cytosol. B. IRF-3 subcellular localization monitored 16 h after SenV infection by immunofluorescence was shown to be predominantly nuclear in cured cells but mostly distributed throughout the cytoplasm in cells containing GBV-B replicon (RepGBNeo5m#15) or in noninfected cells (not shown). Nuclei were counterstained with 4′6′-diamidino-2-phenylindole (DAPI). C. cB76 cell lines containing GBV-B replicon RepGBNeo5m or corresponding IFN-cured cells were cotransfected with pIFN-β-Luc (left) or pISG56-Luc (right) and pCMV-βGal plasmids. Twenty-four hours later, cells were mock infected (empty bars) or infected with SenV at 100 HAU/ml (solid bars) for 16 h prior to lysis for a reporter assay of promoter activities.

FIG. 7.

RIG-I is an intracellular sensor of GBV-B RNA. A. Huh7 cells in 48-well plates were cotransfected with ISG56-Luc and pCMVβgal (50 ng of each), along with 200 ng of vector, RIG-I, or RIG-I C. At 24 h later, cells were mock transfected (DMRIE-C only) or transfected with 2 μg of in vitro-transcribed GBV-B genomic RNA (GBB RNA) or poly(I-C) (pIC) for 20 h before harvest for the reporter gene assay. Relative luciferase activity represents ISG56 promoter activity. B. Huh7.5 cells grown in six-well plates were transfected with 2 μg of the empty vector (lanes 1 through 3), a Flag-tagged WT RIG-I (lanes 4 through 6), or a Flag-tagged mutant RIG-I (KA; lanes 7 through 9). At 48 h posttransfection, cells were mock treated (lanes 1, 4, and 7) or transfected with GBV-B RNA (lanes 2, 5, and 8) or infected with Sendai virus for 20 h (lanes 3, 6, and 9) before cell lysis for immunoblot analysis of ISG56, RIG-I (using an anti-Flag antibody), Sendai virus, and actin (loading control).

FIG. 8.

Alignment of peptide junctions present in viral polyproteins of GBV-B and HCV and cleaved in trans by their respective serine protease with junctions present within MAVS from various species that are cleaved (or predicted to be cleaved) by GBV-B NS3/4A. Residues that flank MAVS cleavage sites by GBV-B NS3/4A and align with natural viral substrates are underlined. Note that the MAVS proteins from various species are different in length. Therefore the positions of (predicted) GBV-B NS3/4A cleavage sites are different.

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GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS - PubMed (original) (raw)