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

This collaborative project aims to define the molecular mechanisms that govern initiation and discontinuous RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The work, supported by an NIH grant (5R21AI171702-02) running from 2022 to 2025, brings together the Borukhov laboratory at Rowan University School of Medicine and the Weiss laboratory at UCLA to reconstitute minimal in vitro systems and apply ensemble and single-molecule approaches to dissect how the viral polymerase initiates RNA synthesis and switches templates to produce subgenomic RNAs. A central objective is to establish determinants that regulate both primer-independent (de novo) and protein-primed initiation of negative and positive RNA strand synthesis. To accomplish this, the teams will assemble an in vitro SARS-CoV-2 transcription/replication system that incorporates the viral RdRp, relevant 5' and 3' UTR RNA elements, and nucleotidylated viral nonstructural proteins nsp8 and nsp9 (uridylated and guanylated). This minimal reconstitution will enable controlled biochemical interrogation of how initiation occurs under defined conditions.

Complementing the reconstitution effort, the project will characterize initiation biochemically and determine the contribution of additional viral protein factors—nsp9, nsp10, nsp13, nsp14—and the nucleocapsid (N) protein to transcription and replication initiation. These studies will use gel-based ensemble assays alongside recently developed single-molecule RdRp activity assays, allowing the investigators to resolve heterogeneous behaviors and transient intermediates that are obscured in bulk measurements. A second major aim is to reconstitute discontinuous transcription in vitro: to model and observe RdRp pausing at transcription-regulating sequences (TRSs), template switching events, and the production of nested subgenomic mRNAs that are characteristic of coronavirus transcription.

To capture template-switching intermediates and the structural states of the polymerase during pausing and backtracking, the researchers will develop both single-molecule and ensemble assays. Planned methods include a single-molecule FRET-based template-switching assay, RNA–protein cross-linking, exonuclease-footprinting, and localized Fe2+-induced hydroxyl-radical mapping. These complementary techniques are intended to define paused, paused-backtracked, and template-switched RdRp complexes at biochemical and structural resolution. The project also seeks to identify viral and human host protein factors required for template switching, thereby linking mechanistic enzymology to potential cellular cofactors of coronavirus transcription.

Collectively, this work is designed to deliver mechanistic insights into how SARS-CoV-2 RdRp initiates RNA synthesis and executes discontinuous transcription, clarifying roles for viral protein cofactors and specific RNA elements. By combining minimal reconstitution with high-resolution biochemical and single-molecule assays, the study aims to reveal transient intermediates and regulatory determinants that underlie coronavirus replication and subgenomic mRNA production. These foundational mechanistic findings could inform downstream efforts to target viral transcription mechanisms therapeutically or to interpret how viral and host factors influence replication fidelity and transcriptome architecture.