Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis
Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis
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DOI：
10.1073/pnas.0508200103
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发表时间：
2006-03-28
影响因子：
11.1
通讯作者：
Ziebuhr, J
中科院分区：
文献类型：
--
作者：
Minskaia, E;Hertzig, T;Ziebuhr, J
严重急性呼吸综合征 (SARS) 冠状病毒 (CoV) 的巨型 RNA 基因组的复制和多达 8 个亚基因组 (sg) mRNA 的合成是由具有高度复杂性的病毒复制酶转录酶介导的，其中包括必需的内切核糖核酸酶活性。在这里，我们表明，与其他 RNA 病毒不同，冠状病毒的复制机制也使用核糖核酸外切酶 (ExoN) 活性，该活性与非结构蛋白 (nsp) 14 相关。SARS-CoV nsp14 的细菌表达形式显示出以 3'-> 5' 方向作用于 ssRNA 和 dsRNA。该活性取决于 DEDD 核酸外切酶超家族中保守的残基。该蛋白质不水解 DNA 或核糖-2'-O-甲基化 RNA 底物，并且需要二价金属离子才能发挥活性。将一系列 5' 标记的 ssRNA 底物加工成大约 8-12 个核苷酸的最终产品。当 dsRNA 的一部分或存在未标记的 dsRNA 时，5' 标记的 RNA 底物被加工成明显更小的产物，表明顺式或反式与 dsRNA 的结合可调节 nsp14 的核酸外切活性。人类 CoV 229E ExoN 活性位点突变体的表征揭示了病毒 RNA 合成的严重缺陷，并且无法回收活病毒。除了基因组复制大幅减少外，还观察到 sg RNA 合成的特定缺陷，例如特定 sg RNA 的大小异常以及各个 sg RNA 物种之间摩尔比的变化。总而言之，该研究确定了一种 RNA 病毒 ExoN 活性，该活性参与从 CoV 的超大基因组 RNA 模板合成多种 RNA。
Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-> 5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximate to 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.