Effect Of MAVS On Classical Swine Fever Virus Infection And Mechanism Of Immunosuppression

Classical swine fever?CSF?caused by classical swine fever virus?CSFV?,is a highly contagious disease of swine that is characterized by high fever,organization hemorrhage and immunosuppression.It results in huge economic losses in the pig industry worldwide.Innate immune response plays an important role as the first line of defense against viruses infection.CSFV inhibits the immune response of host cells to maintain its persistent infection.Therefore,the further study of the interaction between CSFV and innate immune response will help to further understand the mechanism that host defends viruses and the immune escape mechanism of CSFV,providing the theoretical basis for the prevention and control of CSFV.Mitochondrial antiviral-signalling protein?MAVS?plays an important role as a joint protein for regulating the innate immune signaling pathway of hosts.RNA helicases retinoic acid-inducible gene I?RIG-I?-like receptors?RLRs?recognize the invading pathogen and transmit the signal to MAVS.MAVS induces innate immune response to defend viruses infection.In this study,we investigated the effect of MAVS-mediated innate immune response on CSFV infection and the mechanism that CSFV inhibits MAVS-mediated innate immune response and the results are as follow:?1?The effect of MAVS on CSFV replication.CSFV infection increased MAVS expression in porcine alveolar macrophages?PAM?,but UV-CSFV did not increase MAVS expression,indicating that the upregulation of MAVS requires CSFV replication.Additionally,N-acetyl-L-cysteine?NAC,an antioxidant?treatment significantly reduced MAVS expression in CSFV-infected cells,indicating that intracellular ROS is involved in MAVS expression in CSFV-infected PAM.Moreover,MAVS-knockdown increased CSFV replication and MAVS overexpression inhibited CSFV replication,revealing that MAVS has antiviral capacity.Further research showed that MAVS-overexpressing supernatants inhibited CSFV replication and increased CSFV-induced interferon-??IFN-??and interleukin-6?IL-6?,indicating that MAVS inhibites CSFV replication by inducing IFN-I and pro-inflammatory cytokine.?2?The mechanism of that CSFV and its N^pro protein inhibit MAVS-mediated innate immune molecules and apoptosis.We detected the cellular antiviral signaling pathway by overexpression of MAVS using lentivirus infection or transient transfection with 3.1-MAVS.MAVS overexpression markedly upregulated interferon regulatory factor 3?IRF3?and nuclear transcription factor-?B?NF-?B?promoter activities,and increased the expression of tumour necrosis factor?TNF?receptor-associated factors?TRAFs?,IFN-I,IFN-?,pro-inflammatory cytokines and interferon stimulated genes?ISGs?.In addition,MAVS induced apoptosis by caspase-mediated pathway and reduced cell viability,indicating that MAVS can induce the innate immune molecules and apoptosis,which can help hosts to defend CSFV infection.However,CSFV infection reduced MAVS-mediated IFN-?,TNF-?and IL-6 and caspase-dependent apoptosis.The further study analyzed the effect of CSFV proteins on MAVS-mediated IFN-?at the mRNA level.The results showed that CSFV N^pro,NS3 and NS4B reduced MAVS-mediated IFN-?mRNA level.CSFV N^pro also reduced MAVS-mediated TNF-?and IL-6 and apoptosis.Moreover,IRF3 knockdown reduced MAVS-mediated IFN-?,TNF-?and IL-6 expression and apoptosis,indicating that CSFV N^proro inhibits MAVS-mediated innate immune molecules and apoptosis through degradation of IRF3.?3?The mechanism of that CSFV NS4B protein inhibits MAVS-mediated IFN-?.We detected the IFN-?level by overexpression of MAVS using lentivirus infection.NS4B overexpression reduced the expression of IFN-?.In additon,NS4B also reduced the expression of IFN-?in PAM by stimulation with poly?I:C?.Further study showed that NS4B reduced MAVS-mediated IFN-?,indicating that NS4B reduced the expression of IFN-?by inhibiting MAVS pathway.The further study analyzed how NS4B inhibited MAVS pathway.NS4B did not alter the MAVS protein expression,but the C-terminal domain of NS4B could interact with MAVS,and inhibited MAVS-mediated IFN-?,suggested that the C-terminal domain of CSFV NS4B interacts with MAVS and hinders the conduction of MAVS signalling pathway,leading to the decrease of downstream IFN-I.?4?The role of MAVS in IL-8 expression induced by CSFV and its NS4A protein.CSFV infection increased IL-8 expression in swine umbilical vein endothelial cell line?SUVEC?,but UV-CSFV did not increase IL-8 expression,indicating that the upregulation of IL-8requires CSFV replication.In addition,MAVS-konckdown reduced CSFV-induced IL-8,indicating that ROS was involved in CSFV-induced IL-8 expression.The reduction of ROS reduced CSFV-induced IL-8 expression and MAVS-induced IL-8 expression,and the knockdown of MAVS reduced CSFV-induced ROS.Taken together,these data suggest that CSFV induces IL-8 expression through MAVS-ROS pathway.Further study indicates that CSFV NS4A is distributed in the whole cell and induces IL-8 expression through enhancing MAVS signaling pathway and promotes CSFV replication.In conclusion,this study illustrates that MAVS plays an important role in host resistance to CSFV infection;CSFV N^pro inhibits MAVS-mediated innate immune molecules and apoptosis through degradation of IRF3;C-terminal domain of CSFV NS4B interacts with MAVS and inhibits MAVS-induced IFN-I;CSFV induces IL-8 production through MAVS-ROS pathway and CSFV NS4A induces IL-8 production through enhancing MAVS signaling pathway.These data provides a novel theoretical basis for further understanding the mechanisms by which CSFV infection induces innate immunity and its suppression.