Zika Virus Circumvents Host Innate Immunity By Targeting The Adaptor Proteins MAVS And MITA

Objective: In recent years,the outbreak of Zika virus(ZIKV)in many countries and regions around the world has brought extraordinary concern.In the ZIKV endemic areas,the proportion of microcephaly in newborns and Guillain-Barre syndrome(GBS)in infected adults has increased significantly,indicating that ZIKV may has severe neurotoxicity.However,there is no vaccine or specific drug to prevent or treat the diseases caused by ZIKV infection.Thus,it is extremely urgent to characterize the pathogenesis of ZIKV.It has been documented that ZIKV failed to activate a quick and robust innate immune response,suggesting that ZIKV can evade the antiviral immune response of host cells through some strategies.Recently,some progress has been made in understanding the immune escape mechanisms of ZIKV.Yet despite these findings,for ZIKV,virus proteins responsible for the immune escape and their molecular mechanisms are not fully elucidated.Moreover,ZIKV strains from African-lineage and Asian-lineage contain inherent differences in genomic sequences,which may result in new and diverse interactions between ZIKV and the host cells,thus showing unique innate immune responses and pathogenicity.Yet compared with the African strains represented by MR766,contemporary Asian strains have been little studied.In this study,the contemporary ZIKV Asian strains SZ-WIV01 from Guangzhou,China in 2016 was used to study the molecular mechanism of ZIKV for immune evasion.Methods: 1.RT-PCR for IFNB m RNA expression and ELISA for IFN? production wereperformed in SZ-WIV01-infected SH-SY5 Y cells to investigate the antagonismeffect of SZ-WIV01 virus strain on the production of IFN?.2.A series of HA-tagged SZ-WIV01 protein plasmids were constructed andoverexpressed in HEK293 T cells respectively to verify their expression ineukaryotic cells by Western Blot,including three structural proteins(C,pr M and E)and seven nonstructural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5)together with the NS2B3 complex.3.The plasmids expressing viral proteins were transfected into HEK293 T cells,respectively.The expression of IFN? and IFN-stimulated genes(ISGs)such as CCL5and ISG15 were detected by RT-PCR and luciferase reporter assays to screen out theviral proteins involved in the immune evasion of SZ-WIV01.4.The plasmids encoding key proteins in the RLRs signal pathway were constructed,including RIG-I,MAVS,MITA,TBK1,IRF3.Their fate in context of SZ-WIV01infection or viral proteins overexpression were systematically evaluated by RT-PCR,ELISA,Western Blot,immunoprecipitation,ubiquitination or immunofluorescenceanalysis for screening out the target proteins that interacted with viral proteins.5.According to the functionality,plasmids expressing the truncated or point mutants ofviral proteins and their target proteins were constructed.Their expression wasverified by Western Blot.Immunoprecipitation assays were performed to explore thespecific binding regions between viral proteins and their target proteins.6.The effects of SZ-WIV01 and its viral proteins on the expression of target proteinswere assessed by RT-PCR and Western Blot analysis.7.Western Blot and ubiquitination experiments were conducted to investigate theeffects of SZ-WIV01 and its viral proteins on posttranslational modifications of thetarget proteins.Results: 1.SZ-WIV01 virus dramatically inhibited the production of IFN? and ISGs for evadingthe host antiviral immune responses.2.The plasmids encoding SZ-WIV01 proteins,key proteins in the RLRs signal pathwayand their mutants were successfully expressed in eukaryotic cells.3.SZ-WIV01 non-structural proteins NS3 and NS2B3 significantly suppressedpoly(I:C)-triggered expression of IFN? and ISGs together with the nucleartranslocation of IRF3 protein.4.NS3 and NS2B3 targeted MAVS and MITA,respectively,for the RLRs signaltransmission inhibition and IFN? antagonism.5.The binding between NS3 and MAVS depended on NS3(aa 181-480)and MAVS(aa1-180)or(aa 361-540),while NS2B3(aa 1-310)was physically associated withMITA(aa 1-379).6.NS3 and NS2B3 specifically downregulated the expression of MAVS and MITA,respectively.Further analysis showed that the expression of MAVS(aa 361-540)wasdiminished when co-transfected with NS3,while MAVS(aa 1-180)and MAVS(aa181-360)withstood this damage;meanwhile,the overexpression of NS2B3significantly impaired the expression of MITA-C(aa 174-379)but had little effecton MITA-N(aa 1-173).7.NS3 and NS2B3 severally inhibited the expressions of MAVS and MITA at proteinlevel but not at the m RNA level.And this inhibition was effectively reversed byMG132,suggesting that this process was primarily mediated by the proteasomepathway.8.NS3 and NS2B3 severally degraded MAVS and MITA mainly by catalyzing theirK48-linked polyubiquitination.9.NS2B3 also impaired K63-linked polyubiquitination of MITA for RLRs pathwaysuppression.Conclusion: Here we demonstrate that contemporary ZIKV strain SZ-WIV01 can downregulate the production of type I IFN and ISGs along with the expression of MAVS and MITA.Mechanistically,NS3 and NS2B3 target MAVS and MITA respectively and catalyze their K48-linked polyubiquitination for degradation.Moreover,NS2B3 impairs poly(I:C)-triggered K63-linked polyubiquitination of MITA,thereby subverting the activation of downstream sensors.Our study reveals an undiscovered mechanism for ZIKV to escape the innate immune response,providing new insights into clinical study of vaccines or effective drugs.