The Mechanism Of Inhibition Of RLR-Mediated Type Ⅰ Interferon Response By Senecavirus A 2B Protein

Senecavirus(SVA)is the only member of the Senecavirus genus in the Picornaviridae family,which has potent oncolytic activity.In 2007,SVA was first identified as the causative agent of porcine idiopathic vesicular disease,and subsequently it was reported to cause vesicular disease in pigs in many countries such as the United States,Canada,Colombia,Brazil,Vietnam and China.Up to now,there is no commercial vaccine available for control of this disease.The virus has evolved rapidly during the transmission,which has increased the risk of the epidemic.Therefore,it is urgent to clarify the pathogenesis of SVA infection and elucidate how SVA regulates host immune response,which will provide insights and direction for development of vaccines against SVA infeciton.In order to determine the potential mechanism of the inhibition of host innate immunity by SVA infection,the SVA proteins that could inhibit type I interferon(IFN)production were identified and the antagonistic mechanisms were explored.Our findings include:(1)SVA non-structural protein 2B inhibits the expression of type I interferon and ISGs by degradation of VISA in the RLR pathway.The expression of type I interferon and ISGs induced by Se V in HEK293 T or PK-15 cells could be inhibited by SVA infection.To explore the mechanism of cellular antiviral response regulated by SVA,we investigated the effect of SVA non-structural proteins on the activation of IFN-β induced by Se V or Poly(I:C),found that 2B protein significantly inhibited the activation of IFN-β promoter and the expression of IFN-β,MXA or ISG15,ISG54,and OAS1.Therefore,we explored how 2B protein regulates host innate immune response.The results showed that IFN-β promoter activation mediated by RIG-I(CARD),MDA-5 and VISA was inhibited by 2B,but did not affect the activation of downstream molecules of VISA.This suggested that 2B targeted VISA or its upstream molecules to block the signal transduction mediated by RLR.The interaction between 2B and various component of RLR pathway was evaluated by performing Co-IP assay,and we determined that 2B only interacted with VISA,and the co-localization of 2B and VISA was verified by performing the indirect immunofluorescence assay.Subsequently,we determined that 2B degraded VISA protein does not depend on apoptosis,VISA oligomerization,and VISA cleaved.Finally,2B induced the degradation of VISA protein,but did not inhibit the m RNA transcription and translation of VISA,and induces the degradation of VISA depend on one of some degradation pathways.(2)SVA 2B regulate the degradation of VISA depend on Caspase-3 and Caspase-9In order to clarify how 2B degrade VISA,the proteasome inhibitor MG-132,lysosomal inhibitor CQ and Caspase inhibitor Z-VAD-FMK were used to evaluate the effect of 2B on VISA in presence of these inhibitors.Results showed that MG-132 and CQ did not affect the degradation of VISA by 2B,but Z-VAD-FMK restored the VISA abundance in the presence of 2B.The Caspase-2 inhibitor Z-VDVAD-FMK,Caspase-3 inhibitor Z-DEVD-FMK,Caspase-6 inhibitor Z-VEID-FMK,Caspase-8 inhibitor Z-IETD-FMK and Caspase-9 inhibitor Z-LEHD-FMK were further used to identify the key molecular protein involved in regulation of VISA degradation.Results showed that treatment with Z-DEVD-FMK and Z-LEHD-FMK restored the protein level of VISA.This indicated that SVA 2B depend on Caspase-3 and Caspase-9 to regulate the degradation of VISA.(3)The 1~48 and 100~128 amino acids regions are the key functional regions in 2B to degrade VISA.To identify the key regions of 2B involving in degradation of VISA,a series of eukaryotic plasmids expressing the truncated mutants of 2B were constructed.The effect of these mutants on VISA expression was investiaged.The results showed that the degradation of VISA was recovered when the 1~28,20~48 or 100~128 amino acids region of 2B was deleted.Similarly,the inhibition of 2B on the expression of IFN-β and downstream ISGs induced by Se V disappeared as well when the 1~28,20~48 or 100~128 amino acids region of 2B was deleted.These indicated that the 1~48 and 100~128 amino acids regions of 2B were responsible for induction of the degradation of VISA and blocking the expression of type I interferon and ISGs.In summary,we revealed for the first time that SVA 2B inhibited the activation of the RLR pathway and induced the degradation of VISA by activating the apoptosis pathway in this study.The 1~48 and 100~128 amino acid regions of 2B were the key functional domains of 2B to induce the degradation of VISA.These results indicate that SVA 2B has an antagonistic function of host innate immunity,and these data provide new theories for clarification of the pathogenesis of SVA.