Processing of open reading frame la replicase proteins nsp7 to nsp10 in murine hepatitis virus strain a59 replication
Processing of open reading frame la replicase proteins nsp7 to nsp10 in murine hepatitis virus strain a59 replication
复制标题
DOI：
10.1128/jvi.00017-07
复制
发表时间：
2007-10-01
影响因子：
5.4
通讯作者：
Baric, Ralph S.
中科院分区：
文献类型：
--
作者：
Deming, Damon J.;Graham, Rachel L.;Baric, Ralph S.
冠状病毒表达开放阅读框1a（ORF 1a）和ORF 1b多聚蛋白，其中16个非结构蛋白（nsp）衍生自所述多聚蛋白。ORF 1a羧基末端的高度保守区域被nsp 5蛋白酶（M-pro）加工成成熟产物，包括nsp 7、nsp 8、nsp 9和nsp 10，这些蛋白具有参与RNA合成的预测或鉴定的活性。尽管复制需要M-pro对ORF 1ab进行连续翻译和蛋白水解加工，但尚不清楚多聚蛋白内的特异性切割事件是否被抑制。我们确定了在小鼠肝炎病毒（MHV）复制过程中对nsp 7至nsp 10蛋白及其加工的需求。通过使用MHV反向遗传学系统，对nsp 7至nsp 10的编码序列进行框内缺失或切除其侧翼M-pro切割位点，并确定对复制的影响。通过分析M-pro加工、RNA转录和生长适应性来表征活病毒。缺失编码nsp 7至nsp 10的任何区域都是致命的。切割位点的破坏是致命的，除了nsp 9-nsp 10位点的破坏，其导致具有减弱的复制的突变病毒。减毒nsp 9-nsp 10切割突变体的传代增加了接近野生型动力学的适应性，而没有回复到能够处理nsp 9-nsp 10的病毒。我们还证实了nsp 7和nsp 8之间存在第二个切割位点。为了确定不同的功能是否可以归因于多蛋白的预加工形式，包括nsp 7至nsp 10，编码nsp 7和nsp 8的基因被重排。突变病毒是不能存活的，这表明未切割的蛋白质可能是复制或蛋白水解加工所必需的。
Coronaviruses express open reading frame la (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (M-pro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by M-pro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking M-pro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of M-pro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In,order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.