Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Department of Microbiology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Nucleic Acids Res. 2019 Jan 10;47(1):299-309. doi: 10.1093/nar/gky1058.
The L proteins of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus (RABV), possess an unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain with a loop structure protruding into an active site cavity of the RNA-dependent RNA polymerase (RdRp) domain. Here, using complementary VSV and RABV systems, we show that the loop governs RNA synthesis and capping during the dynamic stop-start transcription cycle. A conserved tryptophan residue in the loop was identified as critical for terminal de novo initiation from the genomic promoter to synthesize the leader RNA and virus replication in host cells, but not for internal de novo initiation or elongation from the gene-start sequence for mRNA synthesis or pre-mRNA capping. The co-factor P protein was found to be essential for both terminal and internal initiation. A conserved TxΨ motif adjacent the tryptophan residue in the loop was required for pre-mRNA capping in the step of the covalent enzyme-pRNA intermediate formation, but not for either terminal or internal transcription initiation. These results provide insights into the regulation of stop-start transcription by the interplay between the RdRp active site and the dual-functional priming-capping loop of the PRNTase domain in non-segmented negative strand RNA viruses.
水疱性口炎病毒（VSV）和狂犬病病毒（RABV）等弹状病毒的 L 蛋白具有非常规的 mRNA 帽结构酶（GDP 多核糖核苷酸转移酶，PRNTase）结构域，该结构域带有一个突出到 RNA 依赖性 RNA 聚合酶（RdRp）结构域活性位点腔中的环结构。在这里，我们使用互补的 VSV 和 RABV 系统表明，该环控制着动态停止-启动转录循环中的 RNA 合成和加帽。在环中鉴定出一个保守的色氨酸残基对于从基因组启动子从头起始合成先导 RNA 和病毒在宿主细胞中的复制至关重要，但对于从基因起始序列从头起始合成 mRNA 或前体 mRNA 加帽的内部从头起始或延伸则不重要。发现辅助蛋白 P 对于末端和内部起始都是必需的。环中色氨酸残基附近的保守 TxΨ 基序对于预 mRNA 加帽过程中形成共价酶-pRNA 中间物的步骤是必需的，但对于末端或内部转录起始则不是必需的。这些结果提供了对非分段负链 RNA 病毒中 RdRp 活性位点与 PRNTase 结构域的双重功能起始-加帽环之间相互作用调控停止-启动转录的深入了解。
