The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
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DOI：
10.1074/jbc.c300328200
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发表时间：
2003-10-10
期刊：
影响因子：
--
通讯作者：
Huang JD
中科院分区：
文献类型：
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作者：
Tanner JA;Watt RM;Chai YB;Lu LY;Lin MC;Peiris JS;Poon LL;Kung HF;Huang JD
来自严重急性呼吸综合征冠状病毒（SARS-CoV）的推定的NTR/解旋酶蛋白被假定在病毒生命周期中发挥许多关键作用，使其成为抗SARS治疗的有吸引力的靶点。我们已经克隆，表达，并纯化了这种蛋白质的N-末端六聚组氨酸融合在大肠杆菌中，其特征在于其解旋酶和NTR的活动。该酶解旋双链DNA，依赖于5′单链突出端的存在，表明活性的5′o 3′极性，这是冠状病毒解旋酶的独特特征。我们提供了第一个定量分析的多核酸结合和NTR活性的巢状病毒解旋酶，使用高通量磷酸盐释放试验，将很容易适应于未来的测试解旋酶抑制剂。所有八种常见的NTPs和dNTPs都被SARS解旋酶在镁依赖性反应中水解，受到单链DNA或RNA的刺激。该酶对ATP、dATP和dCTP的偏好优于其他NTP/dNTP底物。同聚核苷酸显著刺激ATP酶活性（15-25倍），但poly（G）和poly（dG）是明显的例外，它们是非刺激性的。我们发现不同的同聚多核苷酸的结合的表观强度有很大的变化，如通过表观Km所观察到的，dT 24的结合比dA 24强10倍以上。
The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km .