NTP介导的猪瘟病毒聚合酶产物剪切机制研究

聚合酶维持其保真度主要通过特异性的底物选择和校正两方面，但RNA病毒编码的RNA依赖的RNA聚合酶（RdRP）曾被认为不具有校正功能。目前有研究表明人类免疫缺陷病毒（HIV）的逆转录酶和丙型肝炎病毒（HCV）的聚合酶具有NTP介导的RNA产物剪切功能，可在病毒基因组复制过程中起到校正的作用，但是其剪切的机制尚不清楚。近期我们通过体外生化实验发现猪瘟病毒（CSFV）的非结构蛋白NS5B（聚合酶）也具有NTP介导的产物剪切功能，同时猪瘟病毒的非结构蛋白NS3可以提高NS5B的剪切效率。在本项目中我们将利用体外酶学实验分析CSFV NS5B剪切的规律，并通过蛋白质晶体学方法揭示NTP介导的聚合酶剪切反应的机制，同时利用体外生物化学、蛋白质晶体学和冷冻电镜等方法来阐明NS3调控NS5B剪切功能的机制。该研究有助于深入了解聚合酶的剪切机制以及NS3与聚合酶之间的调控机制，为抗病毒研究提供科学依据。

Polymerases maintain their fidelity mainly through specific substrate selection and proofreading, but RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses were once considered to have no proofreading function. Previous studies have shown that the HIV reverse transcriptase and HCV RdRP can use NTPs to excise the terminal nucleotide in nascent RNA, suggesting that they may play a role of proofreader during viral genome replication, but the precise mechanism is still unclear. Recently, we found that the nonstructural protein NS5B of classical swine fever virus (CSFV) has the NTP-mediated nucleotide excision activity, and the nonstructural protein NS3 of CSFV improves the excision efficiency of NS5B. We will reveal the nucleotide excision mechanism of CSFV NS5B by in vitro biochemical approaches and protein crystallography. Meanwhile, we will investigate how NS3 regulates NS5B excision function through in vitro biochemical assays, protein crystallography and cryo-electron microscopy. This will contribute to a deeper understanding of the nucleotide excision mechanism of polymerase and the regulation mechanism of NS3 and polymerase, providing a scientific basis for antiviral studies.