ASFV毒力蛋白MGF360-9L降解宿主蛋白MATR3促进病毒复制的研究

Virulence protein MGF360-9L of African swine fever virus protects viral replication in vitro by degrading MATR31

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染所引起的一种高度致死的传染性疾病。ASFV多基因家族编码的MGF360-9L已被证实是ASFV重要的毒力蛋白,是ASFV逃逸宿主天然免疫应答的关键之一。本实验室的前期研究基础表明,宿主细胞核基质蛋白3(Matrin 3,MATR3)可能是MGF360-9L的相互作用蛋白之一,但这种相互作用未经验证。此外,MGF360-9L和MATR3之间是否存在相互调控,对于病毒复制或宿主免疫有何意义尚不可知。因此,本研究首先通过免疫共沉淀(Co-immunprecipitation,Co-IP)验证MGF360-9L与MATR3的相互作用;通过实时荧光定量PCR(RT-qPCR)、蛋白免疫印迹(Western-blot)分析ASFV野生型病毒株(ASFV-WT)、ASFV MGF360-9L缺失毒株(ASFV-ΔMGF360-9L)感染或MGF360-9L过表达对MATR3表达的影响;通过RT-qPCR、Western-blot以及分析ASFV荧光重组毒株复制产生的荧光强度去探究外源过表达MATR3对不同ASFV毒株体外复制的影响。结果,在PK-15细胞中,MGF360-9L与MATR3特异性相互作用并剂量依赖性降解MATR3;在MA-104细胞中,外源过表达MATR3显著抑制ASFV-ΔMGF360-9L体外复制,但不影响ASFV-WT体外复制。本研究证实ASFV毒力蛋白MGF360-9L通过降解宿主限制因子MATR3促进ASFV复制,拓展了MGF60-9L的宿主免疫逃逸途径,为进一步探究ASFV的致病机制奠定了基础。

African swine fever(ASF)is a highly lethal infectious disease caused by African swine fever virus(ASFV) infection.MGF360-9L,encoded by the ASFV multigene family,is an important virulence protein for ASFV escaping host immune response.Past studies had suggested that host protein matrix 3(MATR3) may be one of the interacting proteins of MGF360-9L,but this interaction has not been veri fied experimentally,and the sig nificance of this inter action to ASFV replication or host immune is unknown.In this study,the interaction between MGF360-9L and MATR3 was verified by co-immunoprecipitation(Co-IP).The influences of wild-type ASFV strain(ASFV-WT),MGF360-9L deleting ASFV strain(ASFV-ΔMGF360-9L) infection and MGF360-9L overexpression on the expression of MATR3 were analyzed by quantitative real-time PCR(RT-q PCR) and western blot.The effects of MATR3 on viral replication of ASFV-WT and ASFV-ΔMGF360-9L strains were investigated by RT-q PCR,Western-blot,and viral fluorescence intensity analysis.The results indicated that MGF360-9L specifically interacted with MATR3 and degraded MATR3 in a dose-dependent manner.In addition,overexpression of MATR3 in MA-104 cells significantly inhibits the ASFV-ΔMGF360-9L replication rather than ASFV-WT replication in vitro.The results confirmed that MGF360-9L promotes ASFV replication by degrading the host restriction factor MATR3 in vitro,which expanded the immune evasion mechanism of MGF60-9L and laid a foundation for further exploration of the pathogenesis of ASFV.