RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
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DOI：
10.1371/journal.ppat.1006803
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发表时间：
2017-12-01
期刊：
影响因子：
6.7
通讯作者：
Fearns, Rachel
中科院分区：
文献类型：
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作者：
Braun, Molly R.;Deflube, Laure R.;Fearns, Rachel
非分段负链RNA病毒的大聚合酶亚基(L)转录病毒mrna并复制病毒基因组。对VSV的研究表明，L蛋白的保守区V （CRV）是capping结构域的一部分。然而，CRV折叠并突出到聚合结构域，这表明它也可能在RNA合成中发挥作用。在这项研究中，呼吸道合胞病毒（RSV） CRV的作用通过单氨基酸取代和小分子抑制剂BI-D进行了评估。利用细胞微基因组和体外生化分析分析其作用。几个氨基酸替换抑制了盖帽全长mRNA的产生，相反导致了长度约为40个核苷酸的短转录物的积累，证实了RSV CRV在盖帽中起作用。此外，所有测试的六种变体在RNA复制方面都有部分或完全缺陷。这是由于聚合酶无法有效地延长启动子区域内的RNA。BI-D还能抑制转录和复制。在这种情况下，启动子区域内的聚合酶延伸活性被增强，使得从启动子转录的小RNA没有被释放，而是被拉长，越过了第一个基因启动信号。这伴随着第一个基因启动信号的mRNA起始量减少和不同长度的异常rna的积累。因此，除了其在mRNA capping中的功能外，保守区V还调节聚合酶的延伸特性，以使高效转录和复制发生。
The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and in vitro biochemical assays. Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur.