Putics Akos, Filipowicz Witold, Hall Jonathan, Gorbalenya Alexander E, Ziebuhr John
Institute of Virology and Immunology, University of Würzburg, Germany.
J Virol. 2005 Oct;79(20):12721-31. doi: 10.1128/JVI.79.20.12721-12731.2005.
Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
冠状病毒约30 kb的正链RNA基因组的复制以及大量亚基因组长度RNA的合成是由复制酶-转录酶介导的，复制酶-转录酶是一种膜结合蛋白复合物，包含几种细胞蛋白和多达16种具有多种酶活性的病毒非结构蛋白（nsps），这些酶活性包括蛋白酶、聚合酶、解旋酶、甲基转移酶和核糖核酸酶活性。为了进一步深入了解复制酶基因编码的功能，我们对冠状病毒X结构域进行了表征，该结构域是nsp3的一部分，并且已被预测为一种ADP-核糖-1''-单磷酸（Appr-1''-p）加工酶。人冠状病毒229E（HCoV-229E）和严重急性呼吸综合征冠状病毒X结构域的细菌表达形式显示出以高度特异性的方式将细胞tRNA剪接的副产物Appr-1''-p去磷酸化为ADP-核糖。该酶对其他几种核苷磷酸没有可检测到的活性。在来自嗜热栖热菌的X结构域同源物AF1521的晶体结构的指导下，通过定点诱变靶向HCoV-229E X结构域的潜在活性位点残基。数据表明，HCoV-229E复制酶多聚蛋白残基Asn 1302、Asn 1305、His 1310、Gly 1312和Gly 1313是该酶活性位点的一部分。对一种缺乏Appr-1''-p酶的HCoV-229E突变体的表征显示，对病毒RNA合成和病毒滴度没有显著影响，并且当突变病毒在细胞培养中传代时未观察到回复为野生型序列的情况。体外保守的X结构域活性的明显可有可无性表明冠状病毒复制酶多聚蛋白已经进化到包括非必需功能。这种新酶活性在体内的生物学意义仍有待研究。
