Department of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea.
Arch Virol. 2012 Nov;157(11):2095-104. doi: 10.1007/s00705-012-1404-x. Epub 2012 Jul 13.
The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn(2+). The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3'-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3'-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.
严重急性呼吸综合征冠状病毒（SARS-CoV）的 RNA 基因组由病毒编码的 RNA 复制酶复制，该酶的关键组成部分是非结构蛋白 12（nsp12）。在本报告中，我们描述了全长重组 SARS-CoV nsp12 RNA 依赖性 RNA 聚合酶（RdRp）的生化特性，该聚合酶能够复制病毒 RNA 模板。纯化的 SARS-CoV nsp12 显示出使用同源多聚 RNA 模板的引物依赖性和引物非依赖性 RNA 合成活性。RdRp 活性严格依赖于 Mn(2+)。nsp12 在没有添加寡核苷酸引物的情况下优先复制非聚合嘧啶 RNA 模板。它还能够从 SARS-CoV 的正链和负链基因组的 3'末端从头开始合成 RNA，分别使用 3'末端 36 和 37 个核苷酸的 RNA。使用全长 nsp12 建立的体外 RdRp 测定系统将有助于理解冠状病毒复制的机制，并有助于开发抗 SARS-CoV 药物。
