Paramyxovirus Ⅴ Protein Antagonism MAVS-mediated Ⅰ Interferon Pathway

Newcastle Disease virus belongs to the family of Paramyxoviridae,subfamily Paramyxovirinae with the single,negative genomic RNA,which causes the death of most species of poultry.NDV genome encodes six viral main structure proteins respectively,of which P genome can encode two nonstructure proteinⅤ and protein W.Retinoic acid induced gene-Ⅰ is the important sensor to identify viral RNA excitation the production of type Ⅰ IFN.MAVS is the adapter protein in innate antiviral immunity by RIG-Ⅰ-like receptors,while Newcastle disease virus infects host cells,it can induce large amounts of interferon,thus inhibiting viral proliferation,and Ⅴ protein encoded by Newcastle disease virus can prevent IFN antiviral,so the interaction between the virus Ⅴ protein and celluLar’s protein MAVS become a new hotspot in recent years.V protein of NDV and other paramyxovirus have similar specific structure,recent studies found that Ⅴ protein encoded by paramyxovirus RNA block the function of IFN antiviral activity,so it is closely related to pathogenicity of the virus.It is reported that Ⅴ protein of Newcastle Disease virus have degradation for MAVS signaling pathways,we transfect paramyxovirus Ⅴ protein through exogenous transfection,to detect its effect on MAVS protein and the generate of IFN-β,the resuLt shows that paramyxovirus Ⅴ protein degradates MAVS protein by ubiquitin-proteasome pathway,thus the virus interfere the production of IFN at the levels of MAVS.Through experiments we also get various MAVS Cloning to study the interaction between virus Ⅴ protein and MAVS,finding the Specific reaction between paramyxovirus Ⅴ protein and MAVS.In short,the experimental study tested and verified the important role of virus Ⅴ protein in antagonize interferon.1.NDV induced the degradation of MAVS,while downstream of IFN signaling pathways still activeThe study we use the NDV and SeV infected HeLa 6,9,12,24 hours before MAVS expression levels was detected,the MAVS also appeared completely degraded in the late viral infection.The experiment also demonstrated a dose-dependent effect of NDV-induced MAVS degradation,and NDV can also degrade exogenous transfected MAVS.In order to verify the cells infected with NDV MAVS activation of downstream signaling pathways,HeLa infected NDV,after 3、6、9、12h,we detected TBK1/p-TBK1/pcbp2/Mavs/p-IRF-3 by westwen-blot,the results showed that NDV infection did not cause p-TBK1,p-IRF-3 early activation,and in the late stage of infection apparent activation.This results shows the activation of IFN by TBK1 and IRF-3 may resuLt in other signaling pathway,it needs more experiments to research.2.MAVS was decreased in Ⅴ protein of NDV Constructed by Ding Yunlei found that NDV Flag-V gene is capable of degrading cellprotein MAVS,to further verify that the Ⅴ protein of NDV play a role in cell MAVS degradation in innate immunity,experiments built by the constructed Flag-V gene transfected HeLa cells and A549 cells,then detected the expression level of MAVS.To prove RIG-MDA5 signaling pathway was interfered by Ⅴ protein of NDV,effect Levels from interferon promoter after transfected different concentration gradients Flag-Ⅴ and Flag-MAVS,detected cells IFN-β produced by the luciferase assay,the results shows that with transfected Flag-Ⅴ concentration increased,IFN-β production levels decreased at the level of MAVS.While transfected with downstream adapter of TLR3,there is no effect of protein Trif.3.Paramyxovirus Ⅴ protein degradated MAVSIn our studay,we constructed the paramyxovirus of SeV and MEV,finding that with transfected Ⅴ of concentration,exogenous transfected MAVS and infected NDV induce the reduction of IFN-β level.To further demonstrate whether NDV Ⅴ protein degradation MAVS,the existing laboratory ZJ1 Ⅴ protein mutants and WT infected HeLa cells,then MAVS was observed by western-blot,the results prove that MAVS protein did not appear degradation with the virus Ⅴ protein-deficient cells,it also confirmed the important role of the virus Ⅴ protein of NDV.4.Virus degradates MAVS via the ubiquitin-proteasome pathwayThe 293T cells was transfected with HA-K48,24 hours later infected NDV which detected by CO-IP,and without NDV infection as a control,observing whether MAVS was degradated via ubiquitination or not,showing that after transfection of HA-K48 infected with NDV,the western-blot resuLt shows MAVS was degradated by ubiquitination and proteasome pathway induced by NDV.As we all known,MAVS protein was degradated via two pathways,one is the proteasome pathway,another pathway is autophagy.To verify the impact of these two pathways after NDV infection,MG132,CQ,E64d/PepstatinA,wortmannin different concentrations infect HeLa to detect the delay of MAVS degradation showed that cells treat with autophagy blocking drugs,but MAVS was degradated still,and the proteasome inhibitor MG132 inhibited the MAVS degradation,which prove that MAVS was degrated by ubiquitin-proteasome degradation pathway.5.Specific reaction between NDV Ⅴ protein and MAVSSome studies have demonstrated PCBP2 interaction with MAVS 180-540 this fragment,it has not been ruled out the degradation of MAVS due to PCBP2,in order to demonstrate the interaction between Ⅴ protein and MAVS segment specific,this experiment in 293 cells were co-transfected NDV Ⅴ protein and MAVS segmentation and Western-Blot detection of specific response interactions.The result shows that NDV Ⅴ protein and cell MAVS proteins react specifically at 360-540 fragment.