Discontinuous L-binding motifs in the transactivation domain of the vesicular stomatitis virus P protein are required for terminal de novo transcription initiation by the L protein.
Discontinuous L-binding motifs in the transactivation domain of the vesicular stomatitis virus P protein are required for terminal de novo transcription initiation by the L protein.
复制标题
水泡性口炎病毒 P 蛋白反式激活结构域中的不连续 L 结合基序是 L 蛋白末端从头转录起始所必需的。
DOI：
10.1128/jvi.00246-23
复制
发表时间：
2023
影响因子：
5.4
通讯作者：
Ogino,Tomoaki
中科院分区：
文献类型：
--
作者：
Gupta,Nirmala;Ogino,Minako;Watkins,DeanE;Yu,Tiffany;Green,ToddJ;Ogino,Tomoaki
水疱性口炎病毒非节段负链RNA病毒的原型--水疱性口炎病毒的磷酸(P)蛋白是核糖核酸聚合酶大片段(L)蛋白的辅助因子，在转录和复制中起着关键作用。然而，P蛋白转录反式激活的确切机制仍然不清楚。在这里，我们利用Anin试管转录系统和P蛋白的一系列缺失突变体，将包含51-104残基的区域定位为反式激活结构域(TAD)，该区域是末端新起始的关键步骤，TAD是合成前导RNA和抗基因组/基因组的初始步骤，与L蛋白一起。定点突变表明，TAD内三个不连续的L结合位点上的保守氨基酸残基对于P蛋白的反式激活活性是必不可少的，或者对维持其全部活性是重要的。重要的是，TAD点突变对基因组3‘端前导区域和内部基因全长前导RNA和mRNAs合成的相对抑制作用与末端新梢起始相似。此外，任何被检查的TAD突变都没有改变从内部基因合成的mRNAs的梯度模式，也没有诱导通读转录本的产生。这些结果表明，这些TAD突变主要影响单条目终止-启动转录的末端新起始，而很少影响其他步骤(例如，延伸、终止、内部起始)。P-TAD区必需或重要氨基酸残基的突变对VSV在宿主细胞中的复制是致命或有害的。属于单毒目的非分段负链RNA病毒的依赖于重要RNA的核糖核酸聚合酶L蛋白需要它们的同源辅因子P蛋白或它们的同源辅因子P蛋白进行基因组转录和复制。然而，这些辅因子蛋白在L蛋白转录和复制过程中调控功能的确切作用尚不清楚。在这项研究中，我们揭示了水泡性口炎病毒P蛋白反式激活区域内的三个离散的L结合基序，是L蛋白介导的末端新生起始所必需的，这是合成前导RNA以及基因组/反基因组的第一步。
The phospho- (P) protein, the co-factor of the RNA polymerase large (L) protein, of vesicular stomatitis virus (VSV, a prototype of nonsegmented negative-strand RNA viruses) plays pivotal roles in transcription and replication. However, the precise mechanism underlying the transcriptional transactivation by the P protein has remained elusive. Here, using anin vitrotranscription system and a series of deletion mutants of the P protein, we mapped a region encompassing residues 51–104 as a transactivation domain (TAD) that is critical for terminalde novoinitiation, the initial step of synthesis of the leader RNA and anti-genome/genome, with the L protein. Site-directed mutagenesis revealed that conserved amino acid residues in three discontinuous L-binding sites within the TAD are essential for the transactivation activity of the P protein or important for maintaining its full activity. Importantly, relative inhibitory effects of TAD point mutations on synthesis of the full-length leader RNA and mRNAs from the 3′-terminalleaderregion and internal genes, respectively, of the genome were similar to those on terminalde novoinitiation. Furthermore, any of the examined TAD mutations did not alter the gradient pattern of mRNAs synthesized from internal genes, nor did they induce the production of readthrough transcripts. These results suggest that these TAD mutations impact mainly terminalde novoinitiation but rarely other steps (e.g., elongation, termination, internal initiation) of single-entry stop-start transcription. Consistently, the mutations of the essential or important amino acid residues within the P TAD were lethal or deleterious to VSV replication in host cells.IMPORTANCERNA-dependent RNA polymerase L proteins of nonsegmented negative-strand RNA viruses belonging to theMononegaviralesorder require their cognate co-factor P proteins or their counterparts for genome transcription and replication. However, exact roles of these co-factor proteins in modulating functions of L proteins during transcription and replication remain unknown. In this study, we revealed that three discrete L-binding motifs within a transactivation domain of the P protein of vesicular stomatitis virus, a prototypic nonsegmented negative-strand RNA virus, are required for terminalde novoinitiation mediated by the L protein, which is the first step of synthesis of the leader RNA as well as genome/anti-genome.