Murine hepatitis virus replicase protein nsp10 is a critical regulator of viral RNA synthesis
Murine hepatitis virus replicase protein nsp10 is a critical regulator of viral RNA synthesis
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DOI：
10.1128/jvi.02805-06
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发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Baric, Ralph S.
中科院分区：
文献类型：
--
作者：
Donaldson, Eric F.;Sims, Amy C.;Baric, Ralph S.
冠状病毒的复制需要将由开放阅读框1a/ab编码的大多聚蛋白进行蛋白水解加工，形成假定的功能性中间产物，并最终产生约15种成熟蛋白质。C末端的开放阅读框1a蛋白nsp10与病毒复制复合物共定位，但其在转录/复制中的作用尚未明确。为了研究nsp10在冠状病毒转录/复制中的作用，在鼠肝炎病毒（MHV）感染性克隆中对预测的功能域中的保守残基或带电氨基酸对/三联体进行丙氨酸替换，并对拯救出的病毒进行突变表型分析。在16个构建的克隆中，拯救出了5种有活性的病毒，3种突变病毒未产生细胞病变效应，但能够合成病毒亚基因组RNA，8种无活性。所有有活性的突变体在生长动力学和整体病毒RNA合成方面均有所降低，这表明nsp10是正链或负链合成的辅助因子。有活性的突变体nsp10 - E2加工新生多聚蛋白的能力受损，因为在感染该病毒的细胞中检测到加工中间产物，而在野生型感染中未检测到。将突变映射到严重急性呼吸综合征病毒nsp10的晶体结构上，确定了一个对突变有抵抗力的中心核心。针对锌指结构域内或附近残基的突变产生了无活性的表型，表明这两个结构域对nsp10的功能和MHV的复制至关重要。所有导致有活性表型的突变都映射到中心核心之外的环上，其特征是RNA合成的全面下降。这些结果表明，nsp10是冠状病毒RNA合成的关键调节因子，可能在多聚蛋白加工中发挥重要作用。
Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually similar to 15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.