Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3' polyadenylation of viral plus-strand RNAs.IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

中文翻译：

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人冠状病毒229E非结构蛋白8相关RNA 3'-末端腺苷酸转移酶活性的鉴定和表征。

冠状病毒非结构蛋白8（nsp8）已被建议具有多种活性，包括非规范模板依赖性聚合酶活性。在这里，我们表征了人类冠状病毒229E（HCoV-229E）nsp8的重组形式，发现该蛋白质具有金属离子依赖性RNA 3'-末端腺苷酸转移酶（TATase）活性，而其他核苷酸并未（或效率很低）转移到单链和（全）双链受体RNA的3'端。使用部分双链RNA，如果相反的（模板）链包含短的5'oligo（U）序列，则观察到非常有效的TATase活性，而对于具有其他均聚或杂聚序列的底物，则检测到的活性很小（如果有）。 5'悬垂。寡聚（U）辅助/模板化的TATase对部分双链RNA的活性已被其他两种冠状病毒nsp8蛋白证实，表明该活性在冠状病毒中是保守的。当将相应的突变引入HCoV-229E基因组时，用Ala取代保守的Lys残基消除了nsp8的体外RNA结合和TATase活性，并导致了不可行的表型，证实这些活性是由nsp8介导的，并且对病毒至关重要复制。在其他实验中，我们获得了证据表明nsp8对腺苷酸具有明显的特异性，并且无法将鸟苷酸掺入RNA产物中，这强烈反对了该蛋白先前提出的模板依赖性RNA聚合酶活性。假设在5'处存在oligo（U）延伸 在冠状病毒负链RNA的末端，人们很容易推测（但有待证实）nsp8介导的TATase活性与病毒正链RNA的3'聚腺苷酸化有关。以前，建议将冠状病毒nsp8蛋白用于模板依赖性RNA聚合酶的活性类似于RNA引发酶，甚至是经典的RNA依赖性RNA聚合酶，而最近的研究表明，nsp8（加nsp7）的基本辅因子功能对于nsp12介导的RNA依赖性RNA聚合酶活性是至关重要的。为了调和早期研究中的矛盾数据，该研究使用了其他控件和蛋白质来重新研究冠状病毒nsp8相关的活性。从三种冠状病毒nsp8蛋白获得的数据提供了证据，表明该蛋白共享金属离子依赖性RNA 3' 模板链中的短oligo（U）拉伸极大地刺激了多腺苷酸化活性。相反，发现nsp8无法选择和掺入合适的（匹配）核苷酸来从杂聚模板和其他同聚模板生产cRNA产物。虽然证实了nsp8在冠状病毒复制中的关键作用，但该研究修正了在没有其他蛋白质的情况下由冠状病毒nsp8蛋白介导的活性清单。