口蹄疫病毒3A蛋白变异改变病毒宿主嗜性的分子机制

Changes (mutation or deletion) would occur in non-structure protein 3A of foot-and-mouth disease virus (FMDV ) when FMDV spread in field and passage in the laboratory, resulting in alteration of viral host range and life cycle. However, knowledge regarding the molecular mechanism that mutations in 3A protein of FMDV alter the viral host range and life cycle is poorly understood, which make it difficult to understand how the mutations in 3A protein vary the viral host range and to effectively protect against FMD. In this study, Co-immunoprecipitation assay was used to screen host proteins which interact differently with 3A protein of different length from primary fetal bovine kidney (FBK) cells infected with FMDV harboring different length of 3A protein. The host proteins were identified by tandem mass spectrometry. The key amino acids of this interaction were confirmed by GST pull-down, co-immunoprecipitation assays. The effect of the different host protein on viral replication was assayed by gene silencing or overexpression. This work reveal the important role of 3A protein on viral life cycle and the molecular mechanism that 3A protein of FMDV influence on viral host range. This working will provide a theoretical basis for further understanding the replication mechanism of picornavirus and developing prevention and control measures of FMDV in future.

口蹄疫病毒（Foot-and-mouth disease virus, FMDV）在田间传播和实验室传代时，会发生非结构蛋白3A基因的不同突变（点突变或缺失），导致病毒宿主嗜性或病毒生活周期的改变，但其机制仍不明了，不仅难以解释3A蛋白基因不同变异会怎样改变口蹄疫病毒的宿主性嗜性，而且不利于对口蹄疫的针对性防控。本项目拟以不同变异长度的3A蛋白基因的口蹄疫病毒感染牛肾原代细胞（FBK）为研究模型，利用免疫共沉淀（Co-IP）技术，从感染的细胞中筛选与不同长度3A蛋白互作的差异宿主蛋白，用串联质谱技术进行鉴定；采用Co-IP和pull-down技术确定互作的关键氨基酸位点；通过基因沉默和过表达实验研究宿主蛋白对病毒增殖的影响，以期阐明这些蛋白分子在病毒生活周期中的重要作用，解释FMDV 3A蛋白影响FMDV宿主嗜性的分子机制，为进一步认识小RNA病毒的复制机理和口蹄疫防控提供理论基础。

口蹄疫病毒（Foot-and-mouth disease virus, FMDV）非结构蛋白3A在病毒的复制、毒力和宿主嗜性方面发挥重要的作用。FMDV非结构蛋白3A基因的不同突变（点突变或缺失），导致病毒宿主嗜性或病毒生活周期的改变，但其机制仍不明了，不仅难以解释3A蛋白基因不同变异会怎样改变口蹄疫病毒的宿主性嗜性，而且不利于对口蹄疫的针对性防控。本项目以不同变异长度的3A蛋白基因的口蹄疫病毒感染牛肾原代细胞（FBK），利用免疫共沉淀技术，从感染的细胞中筛选与不同长度3A蛋白互作的差异宿主蛋白，并对其进行了生物信息学分析；挑选波形蛋白验证其与FMDV 3A蛋白的互作，并确定了FMDV 3A蛋白上第15-21位氨基酸负责与波形蛋白的互作；利用反向遗传技术验证了FMDV 3A蛋白与波形蛋白互作对FMDV的存活至关重要；通过波形蛋白过表达和干扰实验发现波形蛋白负调控FMDV的复制；通过丙烯酰胺破坏波形蛋白网络结构发现FMDV的复制依赖于完整的波形蛋白网络。本研究为阐明FMDV的复制机理提供了理论基础。