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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。
Abstract：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.
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基本信息:
DOI：10.13417/j.gab.041.002089
中图分类号:R363
引用信息:
[1]吴梦,齐水水,徐雯等.果蝇原肌球蛋白2抗果蝇C病毒作用研究[J].基因组学与应用生物学,2022,41(Z2):2089-2098.DOI:10.13417/j.gab.041.002089.
基金信息:
国家自然科学基金项目(31871927)资助