鸭坦布苏病毒2'-O甲基转移酶缺陷减毒机制研究

病毒蛋白翻译途径是帽子依赖性翻译过程，黄病毒NS5蛋白是黄病毒属中最大最保守的非结构蛋白，2'-O甲基转移酶位于NS5氮端，其甲基化可使病毒RNA形成与宿主细胞mRNA一样的帽子结构，逃避宿主天然免疫系统的识别和清除，抑制该酶甲基化可降低病毒蛋白翻译，导致病毒毒力显著减弱，现已成为备受关注的抗病毒药物研究方向。鸭坦布苏病毒属于黄病毒属病毒，是近年来新发现、对水禽养殖业造成严重危害的病原之一，然而该病毒NS5蛋白2'-O甲基转移酶的研究尚未见报道。本项目拟建立鸭坦布苏病毒2'-O甲基转移酶体外活性分析方法，继而采取定点突变，筛选酶活性关键氨基酸位点，再以鸭坦布苏病毒全长感染性克隆为基础，通过点突变构建并拯救2'-O甲基转移酶关键氨基酸位点缺陷型鸭坦布苏重组病毒；进一步通过探究重组病毒生物学特性，深入阐释重组病毒抑制帽子结构甲基化的靶向减毒机制，为鸭坦布苏疫病减毒活疫苗的研发提供新策略。

Viral protein depends on the mRNA-capping process for translation. In the genus Flavivirus, NS5 protein is regarded as the largest and most conservative nonstructural protein. Methylation of the 2'-O methyltransferase that located at the N-terminus of NS5 protein will promote viral RNA forming similar cap structure of host mRNA for immune escape, while inhibition of the 2'-O methyltransferase methylation can reduce the translation of viral proteins, resulting in a significant reduction in virulence. Now, it has become a research focus of antiviral drugs. As a newly identified member of Flavivirus, Duck Tembusu Virus (DTMUV) can cause serious harm to waterfowl industry. However, there was no report about the 2'-O methyltransferase of DTMUV NS5 protein yet. In this project, we plan to establish an enzyme activity analysis assay in vitro for screening the key activity sites of DTMUV 2'-O methyltransferase. Then, the key sites defective recombinant of 2'-O methyltransferase will be constructed and rescued based on full-length DTMUV infectious clone. Further, the biological characteristics.determination of the cap structure methylation inhibited recombinants will be carried out for in-depth interpretation of the targeted attenuation mechanism. We believe the results will provide a new strategy for developing DTMUV attenuated live vaccine.