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

点击翻译按钮获取中文摘要

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.