The Research On Mechanism Of Newcastle Disease Virus V Protein Inhibitsinnate Immune By Targeting MAVS

Newcastle disease virus(NDV)is a single-stranded,nonsegmented,negative-sense RNA virus which belongs to Avulavirus inParamyxoviridae.The genome of NDV encodes 6 structural protein and 2 nonstructural protein(V and W).Similar to other viruses,NDV establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell.Among various pattern recognition receptors(PAMPs)of the host,the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein(MAVS),which regulates cellular interferon response.In the present study,we confirmed the interactions of NDV V protein with host cellular protein MAVS,and analyzed biological significances of the interactions.The molecular mechanisms about V protein inhibits innate immune by targeting MAVS.In this study,NDV Herts / 33,ZJ-1,Lasota strains were used to infect HeLa cells.We found that MAVS was degraded by proteasome pathway at the late stage of viral infection without affecting the transcription level of MAVS.We showed that NDV infection induces MAVS degradation in a strain-independent but time-dependent manner.Further analysis showed that different from another viral protein,NDV V protein inhibits the induction of type I interferon(IFN)by MAVS degradation through ubiquitin-proteasome pathway,but another viral protein do not.After virus infection for 12 h,V protein was also found to co-localize with MAVS in the cytoplasm by confocal microscopy analysis.The interaction of V protein with full length and segmentation MAVS was demonstrated by Co-IP and IFA.The interaction ofV protein andthe 360-510 amino acid region of MAVS was determined by Co-IP.We confirmed that V protein can increase ubiquitin level of MAVS.Luciferase reporter assay was performed to inhibit the effect of V protein on IFN-β and ISRE responsive promoter by targeting MAVS.The results showed that V protein could do not target downstream of TBK-1,IKK and IRF5 D to inhibit the IFN-β and ISRE promoter in a dose dependent manner.The lysine ubiquitination site of MAVS was predicted by software.K362 A,K371A,K420 A,K461A and K500 A of MAVS mutant plasmids were constructed.293 T cell lines co-transfected with the mutation of MAVS and V protein expressing plasmids.We found that Lysine at positions 362 and 461 is a key site for L protein-mediated MAVS ubiquitination was demonstrated by Co-IP.Finally the recombinant V-deficient NDV was generated by reverse genetics.Compared with the significant degradation of MAVS by wide type virus,V-deficient NDV infection only induced slight MAVS degradation.In addition,V-deficient NDV induced higher expression of IFN-β and interferon simulated genes at the late stage of infection.Insummary,the present study highlights confirmed that the molecular mechanisms about V protein inhibits innate immune by targeting MAVS,andclarified thatvirus can use a variety of strategies to escape the antiviral response of innate immune.