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

摘要/摘要 这个合作项目的广泛目标是了解启动和启动的分子机制 冠状病毒SARS-CoV-2依赖RNA聚合酶(RdRp)的不连续RNA合成 建立规范这些过程的决定因素。为此，谢尔盖·博鲁霍夫博士在 罗文大学SOM和加州大学洛杉矶分校的Shimon Weiss博士将：(I)重建体外最小的SARS-CoV2 转录/复制系统能够不依赖于引物(从头开始)和蛋白质启动(-)和 ()使用病毒RdRp、5‘-非编码区和3’-非编码区RNA元件以及核苷化(尿苷化)合成核糖核酸链 和鸟苷化)病毒非结构蛋白nsp8和nsp9；(Ii)表征病毒 转录/复制的生物化学启动，并确定病毒蛋白因子(nsp9， NSP10、NSP13、NSP14和N蛋白)，使用基于凝胶的系综分析以及最近开发的单个 分子RdRp活性测定；(Iii)重建SARS-CoV-2不连续转录系统 RdRp在转录调节序列(TRSS)的RNA合成过程中暂停并切换RNA 模板，以产生嵌套的亚基因组mRNAs；(Iv)开发单分子和整体 水平分析，如基于单分子FRET的模板切换分析，RNA-蛋白质交联， 核酸外切酶足迹和Fe2+诱导的羟基自由基定位研究 不连续的转录中间体(例如，暂停、暂停-回溯和模板切换的RdRp (V)确定模板切换所需的病毒和人类宿主细胞蛋白因子。

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.