Structure-Function Analysis of Severe Acute Respiratory Syndrome Coronavirus RNA Cap Guanine-N7-Methyltransferase
Structure-Function Analysis of Severe Acute Respiratory Syndrome Coronavirus RNA Cap Guanine-N7-Methyltransferase
复制标题
严重急性呼吸系统综合症冠状病毒RNA帽鸟嘌呤-N7-甲基转移酶的结构-功能分析
DOI：
10.1128/jvi.00061-13
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发表时间：
2013-06-01
影响因子：
5.4
通讯作者：
Guo, Deyin
中科院分区：
文献类型：
--
作者：
Chen, Yu;Tao, Jiali;Guo, Deyin
冠状病毒在病毒基因组RNA和亚基因组RNA的5'端具有一种帽结构，它是通过病毒编码的鸟嘌呤 - N7 - 甲基转移酶（N7 - MTase）和2' - O - 甲基转移酶（2' - O - MTase）连续甲基化产生的。冠状病毒的N7 - MTase因其与冠状病毒非结构蛋白14（nsp14）所含的一种外切核糖核酸酶（ExoN）存在物理连接而独具特色。在本研究中，通过对严重急性呼吸综合征冠状病毒（SARS - CoV）nsp14进行缺失突变和定点诱变，分析了N7 - MTase的结构 - 功能关系。结果表明，ExoN结构域与N7 - MTase的活性密切相关，这表明冠状病毒N7 - MTase不同于所有其他病毒的N7 - MTase，其他病毒的N7 - MTase可与位于同一多肽中的其他结构域分离。经鉴定对N7 - MTase至关重要的12个关键残基中有2个位于核心ExoN结构域的N末端，这进一步证实了ExoN结构域在nsp14的N7 - MTase活性中的作用。其他10个关键残基分布在整个N7 - MTase结构域，但主要位于S - 腺苷 - L - 甲硫氨酸（SAM）结合口袋以及nsp14的MTase折叠的关键结构元件中。序列基序DxGxPxA（氨基酸[aa]331至338）被确定为SAM结合位点的关键部分。这些结果为了解冠状病毒nsp14 N7 - MTase的结构和功能机制提供了见解。
Coronaviruses possess a cap structure at the 5' ends of viral genomic RNA and subgenomic RNAs, which is generated through consecutive methylations by virally encoded guanine-N7-methyltransferase (N7-MTase) and 2'-O-methyltransferase (2'-O-MTase). The coronaviral N7-MTase is unique for its physical linkage with an exoribonuclease (ExoN) harbored in nonstructural protein 14 (nsp14) of coronaviruses. In this study, the structure-function relationships of the N7-MTase were analyzed by deletion and site-directed mutagenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp14. The results showed that the ExoN domain is closely involved in the activity of the N7-MTase, suggesting that coronavirus N7-MTase is different from all other viral N7-MTases, which are separable from other structural domains located in the same polypeptide. Two of the 12 critical residues identified to be essential for the N7-MTase were located at the N terminus of the core ExoN domain, reinforcing a role of the ExoN domain in the N7-MTase activity of nsp14. The other 10 critical residues were distributed throughout the N7-MTase domain but localized mainly in the S-adenosyl-L-methionine (SAM)-binding pocket and key structural elements of the MTase fold of nsp14. The sequence motif DxGxPxA (amino acids [aa] 331 to 338) was identified as the key part of the SAM-binding site. These results provide insights into the structure and functional mechanisms of coronaviral nsp14 N7-MTase.