Zhang Pengwei, Xie Jian, Yi Guanghui, Zhang Chuyu, Zhou Rong
Institute of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, PR China.
Virus Res. 2005 Sep;112(1-2):9-23. doi: 10.1016/j.virusres.2005.03.003. Epub 2005 Apr 15.
Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.
经典猪瘟病毒（CSFV）非结构蛋白5B（NS5B）编码一种RNA依赖性RNA聚合酶（RdRp），该酶是一种关键酶，可通过无引物的从头合成机制启动RNA复制，是抗病毒治疗的潜在靶点。我们在大肠杆菌中表达了NS5B蛋白。rGTP可刺激RNA合成的从头起始，而GDD基序突变为甘氨酸-天冬氨酸-天冬氨酸（GAA）会消除RNA合成。为了更好地理解CSFV中病毒RNA合成的机制，基于与几种病毒RdRps的比对，通过同源建模构建了三维模型。该模型包含605个折叠成特征性指状、掌状和拇指结构域的残基。指状结构域包含一个在构象变化中起重要作用的N端区域。我们提出，实验观察到的rGTP对聚合酶效率的促进作用可能是由于rGTP结合导致聚合酶构象变化所致。GDD突变为GAA会干扰聚合酶活性位点的残基与金属离子之间的相互作用，从而使聚合酶失活。
