The Mechanism Of EIAV Gag Targeting Mitochondrial Antiviral Signaling Protein MAVS

EIAV can cause acute and severe infectious diseases in equine animals,and the affected horses show fever,jaundice,anemia.A large number of spleen cells were found in necropsy.Many studies have been conducted on the pathogenesis of EIAV-related diseases,but lacking further research on the cellular innate immunity.RIG-I and MDA5 act as receptors for the recognition of exogenous double strands(ds RNA)in cells,and transduce signals to downstream signaling molecules such as MAVS to activate type I interferon(IFN)against viruses.To investigate whether EIAV affects MAVS mediated IFN activation,and IFN-? promoter system was used to confirm that EIAV can regulate the expression of eq MAVS and inhibits the production of IFN.To confirm the role of EIAV direct on eq MAVS expression,we transfected eq MAVS with different doses of EIAV-infected clone plasmid CMV3-8 in HEK293 T cells.Then,we found that EIAV regulated the protein expression of eq MAVS in a dose-dependent manner with co-transfection time significantly.To verify that EIAV also down-regulates endogenous eq MAVS in equine cells,mouse monoclonal antibodies against eq MAVS were prepared in this study,which can detect endogenous eq MAVS in equine cells NBL-6 and e MDM.Using this antibody,we confirmed that EIAV indeed down-regulates endogenous eq MAVS in equine cells.In order to screen out EIAV proteins that interact with MAVS,five EIAV protein plasmids expressing in HEK293 T cells along with the eq MAVS expression constructs.Western blot showed that EIAV Gag could significantly down-regulate the expression of eq MAVS.Fluorescence quantitative assay confirmed that EIAV Gag did not affect the m RNA level of eq MAVS.The localization of eq MAVS and EIAV Gag in cells at different transfection time points was analyzed by laser confocal experiments.It was found that eq MAVS and EIAV Gag had the same intracellular localization in the early stage,and were dispersed in the cytoplasm,but gradually accumulated in mitochondria over time,and no significant colocalization with EIAV Gag was found.The interaction between the two proteins was farther confirmed by PLA and immunoprecipitation.We further explored how EIAV Gag functions,and found that the proteasome inhibitor MG132 could restore the expression of eq MAVS,while EIAV Gag could increase the ubiquitin level of eq MAVS,which confirmed that EIAV Gag degrades eq MAVS through the proteasome pathway.Several E3 ubiquitin ligase was cloned and expressed,and it was found that MUL1 could promote the degradation of eq MAVS by EIAV Gag,and could induce co-immunoprecipitation with EIAV Gag.Further analysis of the main functional domains of EIAV Gag mediated degradation revealed that P15 and P26 could interact with eq MAVS and degrade eq MAVS protein.To our knowledge,the study show that EIAV can regulate MAVS mediated activation of IFN-? and we also produce monoclonal antibodies against eq MAVS to validate the degradation of endogenous eq MAVS by EIAV successfully.The EIAV Gag protein interacting with eq MAVS was further screened.We also found that it recruited the E3 ubiquitin ligase MUL1 to degrade eq MAVS through the ubiquitinproteasome pathway.This research provides an effective biological tool for the exploration of endogenous eq MAVS changes,expands the research on the regulation of MAVS proteins by lentiviral family members,and lays an important foundation for the further study of the signaling pathways of EIAV and RIG-IMAVS,and provides a new research direction for the further exploration of the mechanism of persistent infection induced by EIAV.