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. 2017 Jul 27;91(16):e00823-17.
doi: 10.1128/JVI.00823-17. Print 2017 Aug 15.

Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage

Suhong Qian  1   2 , Wenchun Fan  1   2 , Tingting Liu  1   2 , Mengge Wu  1   2 , Huawei Zhang  1 , Xiaofang Cui  1   2 , Yun Zhou  1   2 , Junjie Hu  3 , Shaozhong Wei  3 , Huanchun Chen  1   2   4 , Xiangmin Li  5   2   4 , Ping Qian  5   2   4
Affiliations

Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage

Suhong Qian et al. J Virol. .

Abstract

Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-β (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3Cpro). SVV 3Cpro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-β. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules.IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3Cpro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for the Toll-like receptor 3 (TLR3)-mediated and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated signaling pathway. We also found that SVV is sensitive to IFN-β. These findings increase our understanding of the interaction between SVV and host innate immunity.

Keywords: 3C-like protease; MAVS; RNA virus; Seneca Valley virus; TANK; TRIF; cell signaling; innate immunity; interferons.

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Figures

FIG 1
FIG 1
SVV infection does not trigger host type I interferon production. (A) Luciferase activity assay from 293T cells infected with Sev (HA titer, 32) or SVV (MOI, 1) for the indicated times. (B) qRT-PCR assay performed with 293T cells infected with Sev (HA titer, 32) and SVV (MOI, 1) for the indicated times. (C) Immunoblot analysis for IRF3 activation in 293T cells infected with Sev (HA titer, 32) or SVV (MOI, 1) for the indicated times. Quantification of the extent of the increase in phosphorylated IRF3 was normalized to total IRF3 (right panel). (D) Immunofluorescence microscopy analysis for the nuclear translocation of IRF3 in 293T cells infected with Sev (HA titer, 32) or SVV (MOI, 1) for 12 h. IRF3 is green and the nucleus is blue. (E) 293T cells were pretreated with IFN-β at the indicated concentration for 12 h and then infected with VSV-green fluorescent protein (GFP) or SVV. CPE was observed at 24 h postinfection. (F) The virus progeny from panel E were determined by plaque assay. Data are shown as means ± SD. ***, P < 0.001. Data are representative of those from at least three independent experiments.
FIG 2
FIG 2
SVV 3Cpro antagonizes Sev-induced type I IFN production. (A) IFN-β and ISG56 mRNA expression as measured by qRT-PCR from 293T cells infected with SVV (MOI, 5) for 3 h and then infected or not with Sev (HA titer, 32) for another 6 and 9 h. (B) Luciferase activity assay of 293T cells transfected with plasmids expressing the indicated SVV protein for 20 h and then infected or not with Sev (HA titer, 32) for another 9 h. Protein expression was detected by Western blotting (bottom). GST, recombinant protein glutathione S-transferase. (C) Luciferase activity assay of 293T cells transfected with increasing levels of SVV 3Cpro-expressing plasmids (50, 100, and 250 ng) for 20 h and then infected or not with Sev (HA titer, 32) for another 9 h. (D) IFN-β and ISG56 mRNA expression as assessed by qRT-PCR from 293T cells transfected with SVV 3Cpro-expressing plasmids or empty vector for 20 h and then infected or not with Sev (HA titer, 32) for another 9 h. (E) Immunoblot analysis of 293T cells transfected with increasing levels of SVV 3Cpro-expressing plasmids (0, 200, and 800 ng) for 20 h and then infected or not with Sev (HA titer, 32) for another 9 h. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of those from at least three independent experiments.
FIG 3
FIG 3
SVV 3Cpro mediates the cleavage of MAVS, TRIF, and TANK. (A) Luciferase activity assay from 293T cells transfected with the indicated adaptor-expressing plasmids or empty vector for 20 h and then infected or not with SVV (MOI, 5) for another 9 h. (B) Luciferase activity assay from 293T cells cotransfected with the indicated adaptors plus SVV 3Cpro or empty vector for 24 h. (C) IFN-β and ISG56 mRNA expression as assessed by qRT-PCR from 293T cells cotransfected with the indicated adaptors plus SVV 3Cpro or empty vector for 24 h. (D) Immunoblot analysis of 293T cells cotransfected with the indicated adaptors (N-terminally Flag tagged) plus SVV 3Cpro or empty vector for 24 h. (E to G) Plasmids expressing Flag-MAVS (E), Flag-TRIF (F), and Flag-TANK (G) were cotransfected with plasmids expressing HA-tagged 3Cpro derived from EMCV (JQ864080) and EV71 (JN230523) into 293T cells. The protein patterns were detected by immunoblotting. (H) Immunoblot analysis of 293T cells infected with SVV (MOI, 5) for the indicated times. Antibodies against MAVS, TANK, and SVV VP1 were used. (I) Immunoblot analysis of 293T cells transfected with Flag-TRIF for 20 h and then infected with SVV (5 MOI) for the indicated times. Antibodies against TRIF and SVV VP1 were used. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of at least three independent experiments.
FIG 4
FIG 4
Protease activity is required for SVV 3Cpro-mediated cleavage of MAVS, TRIF, and TANK. Plasmids expressing Flag-MAVS (A), Flag-TRIF (B), and Flag-TANK (C) were cotransfected with plasmids expressing SVV 3Cpro or empty vector into 293T cells. The inhibitors MG132 (10 μM), NH4CL (10 mM), and z-VAD-FMK (50 μM) were added as indicated at 12 h posttransfection and for another 12 h. The protein patterns were assessed by immunoblotting. (D) Amino acid sequence alignment of 3Cpro derived from SVV (APA28975), FMDV (NP_74046), EMCV (NP_740410), HAV (AKI05745), and EV71 (AFP66570). (E to G) Plasmids expressing Flag-MAVS (E), Flag-TRIF (F), and Flag-TANK (G) were cotransfected with plasmids expressing wild-type SVV 3Cpro and indicated 3Cpro mutations for 24 h. The protein patterns were assessed by immunoblotting.
FIG 5
FIG 5
SVV 3Cpro interacts with MAVS, TRIF, and TANK. 293T cells were transfected with Flag-MAVS and empty vector, HA-3Cwt, or HA-3Cdm for 24 h. Subsequently, the interaction between MAVS and 3Cpro was measured by anti-Flag immunoprecipitation (A). (B) Anti-Flag immunoprecipitation for the interaction between Flag-TRIF, HA-3Cwt, and HA-3Cdm. (C) Anti-Flag immunoprecipitation for the interaction between Flag-TANK, HA-3Cwt, and HA-3Cdm. WCL, whole-cell lysate.
FIG 6
FIG 6
SVV 3Cpro cleaves MAVS, TRIF, and TANK at specific sites. (A) SVV polyprotein organization and the proposed SVV 3Cpro cleavage sites (red). The indicated viral proteins were colored. (B) (Top) Schematic representation of Flag-MAVS and its mutants. (Bottom) 293T cells transfected with the indicated Flag-MAVS construct and empty vector or HA-3Cwt were lysed and subjected to immunoblot analysis. CARD, caspase recruitment domain; TM, transmembrane. (C) (Top) Schematic representation of Flag-TRIF and its mutants. (Bottom) 293T cells transfected with the indicated Flag-TRIF construct and empty vector or HA-3Cwt for were lysed and subjected to immunoblot analysis. NTD, N-terminal domain; TIR, Toll/interleukin-1 receptor domain; SIA, sufficient to induce apoptosis. (D) (Top) Schematic representation of Flag-TANK and its mutants. (Bottom) 293T cells transfected with the indicated Flag-TANK construct and empty vector or HA-3Cwt for were lysed and subjected to immunoblot analysis. TBD, TRAF binding domain; ZF, zinc finger.
FIG 7
FIG 7
Cleaved MAVS, TRIF, and TANK lose their functions to trigger type I IFN production. (A) Luciferase activity assay of 293T cells transfected with wild-type and Q148A mutant MAVS plus empty vector, HA-3Cwt, or HA-3Cdm. (B) IFN-β expression as determined by qRT-PCR from 293T cells transfected with wild-type and Q148A mutant MAVS plus empty vector, HA-3Cwt, or HA-3Cdm. (C and D) The effects of cleavage fragments of MAVS by SVV 3Cpro on MAVS-mediated IFN-β production were assessed by IFN-β reporter assay (C) and qRT-PCR assay (D). (E) Luciferase activity assay of 293T cells transfected with wild-type and Q159A mutant TRIF plus empty vector, HA-3Cwt, or HA-3Cdm. (F) IFN-β expression as determined by qRT-PCR of 293T cells transfected with wild-type and Q159A mutant TRIF plus empty vector, HA-3Cwt, or HA-3Cdm. (G and H) The effects of cleavage fragments of TRIF by SVV 3Cpro on TRIF-mediated IFN-β production were assessed by IFN-β reporter assay (G) and qRT-PCR assay (H). (I) Luciferase activity assay of 293T cells transfected with wild-type and E272A-Q291A mutant TANK plus empty vector, HA-3Cwt, or HA-3Cdm. (J) IFN-β expression as determined by qRT-PCR of 293T cells transfected with wild-type and E272A-Q291A mutant TANK plus empty vector, HA-3Cwt, or HA-3Cdm. (K and L) The effects of cleavage fragments of TANK by SVV 3Cpro on TANK-mediated IFN-β production were assessed by IFN-β reporter assay (K) and qRT-PCR assay (L). Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of those from at least three independent experiments.
FIG 8
FIG 8
SVV 3Cpro-mediated TANK cleavage facilitates the TRAF6-NF-κB signaling pathway. (A) NF-κB reporter assay of 293T cells transfected with Flag-TRAF6 and Flag-TANK, HA-3Cdm, or HA-3Cwt for 24 h. (B) Endogenous transcription of TNF-α as determined by qRT-PCR from 293T cells transfected with Flag-TRAF6 and either Flag-TANK, HA-3Cdm, or HA-3Cwt. (C) Immunoblot analysis for IκBα from 293T cells transfected with Flag-TRAF and empty vector, Flag-TANK, HA-3Cdm, or HA-3Cwt for 24 h. (D) NF-κB reporter assay from 293T cells transfected with Flag-TRAF6 and empty vector, Flag-TANKwt, Flag-TANKdm, or HA-3Cwt for 24 h. (E) NF-κB reporter assay from 293T cells transfected with Flag-TRAF6 and empty vector, Flag-tagged wild-type TANK, and the N291 fragment of TANK for 24 h. (F) 293T cells transfected with empty vector or amounts increased wild-type SVV 3Cpro for 20 h and then infected or not with Sev (HA titer, 32) for another 9 h. The cells were lysed and subjected to immunoblot analysis. (G) Endogenous transcription of TNF-α and IL-6 as measured by qRT-PCR from 293T cells infected with SVV (MOI, 5) at the indicated time points. IκBα and VP1 protein expression was detected by Western blotting (bottom). Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of those from at least three independent experiments.
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