果蝇原肌球蛋白2抗果蝇C病毒作用研究

Investigation of the Antiviral Role of Tropomyosin 2 Against Drosophila C Virus in Drosophila melanogaster

黑腹果蝇(Drosophila melanogaster)是研究宿主与病毒互作机制的模式生物,RNA干扰(RNA interference,RNAi)是果蝇最主要的抗病毒机制。果蝇C病毒(Drosophila C virus,DCV)是天然感染果蝇的正链RNA病毒,通过编码RNA沉默抑制子(viral suppressors of RNAi,VSR)1A蛋白(DCV1A)来抑制果蝇RNAi通路。本研究以DCV1A为诱饵蛋白,通过免疫共沉淀(co-immunoprecipitation,Co-IP)和质谱分析,获得了与DCV1A可能存在互作的108个靶蛋白。通过免疫共沉淀和Western blot分析,在果蝇S2细胞中验证了DCV1A与原肌球蛋白2(tropomyosin 2,TM2)存在互作。当细胞感染DCV后,细胞内Tm2的表达水平被显著诱导上调;通过RNAi干扰细胞中Tm2的表达后DCV复制水平显著增加,过表达Tm2基因后DCV复制水平有所下降。在S2细胞中共表达DCV1A和Tm2基因时,DCV1A蛋白水平显著下调,DCV复制也受到抑制。在Tm2突变型果蝇和野生型果蝇的胸腔内注射DCV,监测存活率发现,与野生型果蝇相比,Tm2基因突变体果蝇对DCV更为敏感。研究结果表明,在果蝇细胞水平和成虫水平上Tm2均发挥抑制DCV复制的作用。

Drosophila melanogaster is an established model to study innate immune responses and host-virus interactions.RNA interference(RNAi)is one of the main antiviral mechanisms of Drosophila.Drosophila C virus(DCV)is a positive-sense RNA virus which is a natural pathogen of Drosophila,it encodes a viral suppressor of RNAi(VSR)DCV1A to counteract the RNAi pathway.In this study,a total of 108 proteins were identified in Drosophila S2 cells that may interact with DCV1A by co-immunoprecipitation(Co-IP)and mass spectrometry.Then the interaction between DCV1A and TM2 was validated by Co-IP and Western blot to identify the interacting protein TM2 of DCV1A.The expression level of Tm2 was increased in S2 cells infected with DCV;When DCV1A and Tm2 genes were co-expressed in S2 cells,the level of DCV1A protein was significantly down-regulated,then DCV replication was also suppressed in the co-transfected cells.Both Tm2 mutant and wild-type fruit flies were intrathoracically injected with DCV and the survival rates were monitored daily.The results showed that Tm2 mutant flies were more susceptible to DCV infection than wild-type fruit flies.Taking together,the results presented in the study demonstrated that Tm2 inhibited DCV replication in both S2 cells and adult fruit flies.