SGER: Structure and Function of the SARS Virus RNA Polymerase

SARS病毒，SCoV，是一种新出现的，高毒性的成员冠状病毒家族的正链RNA病毒。 虽然冠状病毒具有RNA复制和转录的独特方面，但这些过程的关键酶RNA依赖性RNA聚合酶（RdRp）的生化和结构信息很少。 该SGER项目将联合收割机结合两位主要研究者的生物化学和结构生物学专业知识：1）利用寡核苷酸构建表达SCoV多伦多分离株RdRp的cDNA，使用常规和亲和层析纯化蛋白，建立聚合酶测定法，鉴定催化残基，并绘制体外RNA合成所需的最小区域; 2）表征由SCoV RdRp引发的RNA合成;以及3）生长SCoV RdRp的高分辨率晶体，并使用X射线晶体学来阐明单独的蛋白质和结合至底物模拟物的蛋白质的结构。 这些研究的结果将建立生物化学测定并解决SCoV聚合酶的结构。 这些结果将与病毒RNA复制的机制高度相关，并将允许设计针对重要类别的人类和动物病原体的更有效的抗病毒药物。

The SARS virus, SCoV, is a newly emerged, highly virulent member of the coronavirus family of positive-strand RNA viruses. While coronaviruses have unique aspects of RNA replication and transcription, little biochemical and structural information exists for the key enzyme of these processes, the RNA-dependent RNA polymerase (RdRp). This SGER project will combine the biochemical and structural biology expertise of the two principal investigators to: 1) use oligonucletoides to construct a cDNA to express the RdRp of the Toronto isolate of the SCoV, purify the protein using conventional and affinity chromatography, establish a polymerase assay, identify the catalytic residues, and map the minimal region required for RNA synthesis in vitro; 2) characterize the initiation of RNA synthesis by the SCoV RdRp; and 3) grow high-resolution crystals of the SCoV RdRp and use X-ray crystallography to elucidate the structure of the protein alone and bound to substrate mimics. Results from these studies will establish biochemical assays and solve the structure for the SCoV polymerase. The results will be highly relevant to the mechanism of viral RNA replication and will allow the design of more effective antivirals against a significant class of human and animal pathogens.