The Effect Of Mitochondrial Protein MAVS On Host Anti-Swine Influenza Virus Infection

Swine influenza is an acute respiratory diseases caused by swine influenza virus.Swine influenza virus is a negative(-)sense ssRNA virus,which enables the dynamic evolution of virus by both antigenic drift and genetic shift to evade host defense.Therefore,there is a critical need to evaluate the immediate response to viral infection and the role of host protein in the innate immune system.1.The effect of swine influenza virus infection on the innate immunity signaling pathways of porcine alveolar macrophagesPorcine alveolar macrophages(PAM)were collected from the lungs of 35-day-old piglets and cultured for 24 h followed by infection with H3N2 swine influenza virus A/Swine/Shandong/3/2005 at a multiplicity of infection(MOI)of 5.Virus proliferation in macrophages was observed by indirect immunofluorescence assay(IFA).Fluorescence quantitative PCR was used to determine the mRNA expression of TLR and RLR signaling pathway related receptors,adapter proteins and effect factors.The data showed that the mRNA of TLR-3,TLR-7,RIG-I and MDA-5 significantly increased at 6-24 hpi(p<0.05);IRF-7 and IFN-?mRNA expression increased significantly at 8-24 hpi(p<0.05);MyD88 and MAVS mRNA increased at 8 hpi and then began a slow decline after 12 hpi.The concentration of TNF-a,IL-1?,IFN-a and IFN-?were measured from supernatants by radioimmunoassay.The results showed that IL-1?and TNF-a peaked at 6 hpi and 8 hpi,respectively,while IFN-a and IFN-?increased significantly from 6-8 hpi(p<0.05).Our findings suggested that swine influenza virus strains could proliferate in PAM and had an influence on the expression of TLR and RLR signaling pathway related genes and proteins.2.The effect of swine influenza virus infection on the innate immunity signaling pathways of epithelial cellsEpithelial cells A549 were infected with SIV at a MOI of 5 and supernatants and cells were collected at different timepoints,respectively.The data showed that swine influenza virus could effectively proliferate in A549 cells with virus titer up to 106.1 TCID50/ml at 48 hpi,which was further confirmed by indirect immunofluorescence assay.TLR and RLR signaling pathway related receptors and adapter proteins were detected by fluorescence quantitative PCR.The results showed that the mRNA of TLR-3,TLR-7,RIG-?and MDA-5 significantly increased from 6 hpi(p<0.05).IRF-7,IFN-?and IFN-?mRNA significantly rose at 8-24 hpi(p<0.05).MyD88 and MAVS mRNA increased at 8 hpi and then decreased from 12 hpi,with no significant difference at 24 hpi(p>0.05).Western blot analysis showed that the expression of RIG-?,TLR-3 and TLR-7 proteins increased while MAVS decreased after SIV challenge.Furthermore,A549 cells stimulated with poly I:C or infected with SIV could activate the luciferase reporter plasmid of pIFN-P-Luc,pIRF3/7-Luc and pNF-KB-Luc.The results illustrated that swine influenza virus could proliferate in A549 cells and had an influence on the expression of TLR and RLR signaling pathway related genes and proteins,which suggested that A549 cells could be used as a model for further research.3.MAVS inhibited the proliferation of swine influenza virus by mediating the production of type ? interferonThree truncation mutants,lacking the CARD-like domain(?CARD),the transmembrane domain(ATM)or the proline-rich domain(AProl),respectively,were constructed based on the pCMV-Flag-MAT-TAG-MAVS plasmid.The results showed that overexpression of full-length MAVS could activate the IFN-?promoter luciferase reporter(pIFN-?-Luc)and inhibit SIV replication in a dose-dependent manner.Overexpression of ACARD and ATM truncation mutants had no effect on the activation of pIFN-?-Luc,pIRF3/7-Luc and pNF-KB-Luc reporters or inhibition of SIV replication.Overexpression of AProl had lower but nonsignificant effect on the activation of pIFN-(3-Luc,pIRF3/7-Luc and pNF-KB-Luc reporters,and could inhibit SIV replication,compared with full-length MAVS(p>0.05).These results indicated that CARD and TM domain were necessary for the function of production of type ? interferon mediated by MAVS.Then,A549 cells were transfected with siRNA to inhibit the endogenous MAVS expression followed by SIV infection,and the result showed that silencing of the endogenous MAVS expression led to an significant inhibition of pIFN-?-Luc and an increase of viral titer(p<0.05).These results demonstrated that MAVS is a pivotal cellular antiviral protein whose expression directly determines antiviral immunity.4.MAVS mediating apoptosis induced by virusThree truncation mutants,lacking the CARD-like domain(ACARD),the transmembrane domain(ATM)or the proline-rich domain(AProl),respectively,were constructed from the pCMV-Flag-MAT-TAG-MAVS plasmid.The results showed that overexpression of full length MAVS could significantly increase apoptosis in a dose-dependent manner(p<0.05).Western blot showed that caspase-3 and caspase-9 protein upregulated significantly in MAVS-overexpression cells(p<0.05).MAVS mutants,with the CARD-like domain or the transmembrane region completely deleted significantly reduced the percentage of apoptosis cells(p<0.05),whereas mutants containing the CARD-like and TM region(AProl)induced apoptosis similar to full-length MAVS.RNA interference detected that transfection of MAVS siRNA oligos into A549 cells significantly reduced the percentage of Annexin V positive cells(p<0.05),which demonstrated that MAVS was essential for H3N2 swine influenza virus induced apoptosis of epithelial cells.Transwell plate analysis demonstrated that MAVS-mediated apoptosis was not a consequence of IFN-I induction by MAVS.The results suggested that MAVS mediated apoptosis was important in antiviral immunity of host.5.The effect of SIV proteins on production of type ? interferon and apoptosis mediated by MAVSNS1,NP and PB1-F2 plasmids were co-expressed with full-length MAVS plasmid in A549 cells to examine whether these proteins were involved in MAVS-mediated apoptosis or production of type ? interferon.The results showed that NS1,PB1-F2 could reduce the activity of the IFN-(3 promoter luciferase reporter(pIFN-?-Luc)and NS1 did in a dose-dependent manner.Of the 3 tested proteins,NS1 significantly altered MAVS-mediated apoptosis of A549 cells in a dose-dependent manner as measured by Annexin V and PI staining(p<0.05),while PB1-F2 or NP had little effect on cell viability compared with MAVS controls(p>0.05).Virus titer was determined by plaque assays.The data showed that NS1,but not PB1-F2 or NP co-expressed with MAVS could inhibit the anti-SIV activity of MAVS and facilitate virus replication.These results suggested that NS1 could antagonize MAVS-mediated apoptosis and the production of type ? interferon.In conclusion,the TLRs and RLRs signaling pathways could be activated after H3N2 swine influenza virus infection.MAVS was essential in the induction of IFN-I and apoptosis.Meanwhile,NS1 of swine influenza virus could antagonize MAVS-mediated apoptosis and the production of type ? interferon.