Untangling the mechanisms of initiation and discontinuous RNA synthesis by COVID-19 RdRp

总结/摘要 这个合作项目的广泛目标是了解启动的分子机制， 冠状病毒SARS-CoV-2 RNA依赖性RNA聚合酶（RdRp）的不连续RNA合成， 确定调节这些过程的决定因素。为此，谢尔盖·博鲁霍夫博士的实验室在 罗文大学SOM和加州大学洛杉矶分校的Shimon韦斯博士将：（i）在体外重建最小的SARS-CoV 2 转录/复制系统，其能够引物非依赖性（从头）和蛋白质引发（-）的起始， （+）使用病毒RdRp、5 '-UTR和3'-UTR RNA元件和核苷酸化（尿苷化）的RNA链合成 和鸟苷酸化）病毒非结构蛋白nsp 8和nsp 9;（ii）表征病毒的过程， 转录/复制起始的生物化学作用并确定病毒蛋白因子（NSP 9， nsp 10、nsp 13、nsp 14和N蛋白），使用基于凝胶的整体测定以及最近开发的单- 分子RdRp活性测定;（iii）重建SARS-CoV-2不连续转录系统，其中 RdRp在RNA合成过程中在转录调节序列（TRS）处暂停， 模板，以产生嵌套的亚基因组mRNA集;（iv）开发单分子和整体 水平测定，例如基于单分子FRET的模板转换测定，RNA-蛋白质交联， 核酸外切酶足迹法和局部Fe 2+诱导的羟基自由基作图来表征 不连续的转录中间体（例如，暂停、暂停-回溯和模板切换的RdRp 复合物）;（V）鉴定模板转换所需的病毒和人宿主细胞蛋白因子。

SUMMARY/ ABSTRACT The broad goal of this collaborative project is to understand the molecular mechanisms of initiation and discontinuous RNA synthesis by the coronavirus SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and establish determinants that regulate these processes. To this end, the laboratories of Dr. Sergei Borukhov at Rowan University SOM and of Dr. Shimon Weiss at UCLA will: (i) reconstitute an in vitro minimal SARS-CoV2 transcription/replication system capable of primer-independent (de novo) and protein-primed initiation of (-) and (+) RNA strand synthesis using viral RdRp, 5’-UTR and 3’-UTR RNA elements, and nucleotidylated (uridylated and guanylated) viral non-structural proteins nsp8 and nsp9; (ii) characterize the process of viral transcription/replication initiation biochemically and determine the contribution of viral protein factors (nsp9, nsp10, nsp13, nsp14, and N protein), using gel-based ensemble assays as well as recently developed single- molecule RdRp activity assays; (iii) reconstitute SARS-CoV-2 discontinuous transcription system in which RdRp pauses during RNA synthesis at transcription-regulating sequences (TRSs) and switches RNA templates to produce nested sets of sub-genomic mRNAs; (iv) develop both single-molecule and ensemble level assays such as single-molecule FRET based template switching assay, RNA-protein cross-linking, exonuclease-footprinting and localized Fe2+ -induced hydroxyl-radical mapping to characterize the discontinuous transcription intermediates (e.g., paused, paused-backtracked, and template-switched RdRp complexes); (v) identify viral and human host cell protein factors that are required for template-switching.