# Structure, function, and inhibition of the SARS-CoV-2 replication-transcription complex

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2021 · $638,072

## Abstract

Project Summary
COVID-19, caused by the coronavirus SARS-CoV-2, continues to devastate the world. In less than a year,
there have been more than 20 million cases with over 700,000 deaths. The viral RNA-dependent RNA
polymerase (RdRp) is the central enzyme responsible for transcription and replication of the viral RNA
genome. This enzyme is also a target for the current antiviral, remdesivir, used to ameliorate the severity and
duration of this disease. The virus also encodes several nucleic acid processing enzymes, in addition to the
RdRp, including a helicase, an endonuclease, an exonuclease, and methyltransferases. However, it is
unknown how these enzymes coordinate to transcribe and replicate the viral genome. This proposal builds
upon preliminary data of the structure of the helicase, nsp13, in complex with the RdRp and a primed substrate
RNA (nsp13-replication/transcription complex or nsp13-RTC). The aims here include completing the structural
analysis of this complex by utilizing additional data collected. The result of this aim will provide higher
resolution (better than 2.7 Å in some parts the RdRp), providing a rich basis for the development of antiviral
inhibitors. Also, having this structure in hand allows for the collaboration with expert developers of
antimicrobials, also part of the aims, including the investigation of the structural details of the pre-incorporation
state of remdesivir and antivirals produced by human microbiome.
The models resulting from the structure of nsp13-RTC serve as foundations to test how the helicase and
exonuclease function together with the RdRp. Specifically, real-time fluorescence assays, single-molecule
fluorescence resonance energy transfer (FRET), and multi-color fluorescence microscopy will be used to probe
the role of the helicase and the exonuclease in unwinding substrate RNA, backtracking, and proofreading.
Another aim applies the pipeline used to characterize the nsp13-RTC assembly, which yielded a high-
resolution structure of the complex, to other RTC assemblies. Specifically, native electrophoretic mobility
assays will be used as a starting point to probe larger assemblies of the RTC. Native mass-spectrometry will
then be used to determine the composition and stoichiometry of the complexes. Finally, cryo-EM will be
applied to solve the structures of these macromolecular machines. The resulting structures will provide a
starting point to elucidate the coordinated functions of these enzymes, provide insight into their mechanisms,
and establish novel targets for therapeutics.
In summary, this proposal aims to understand at the molecular and structural level how the SARS-CoV-2
nucleic acid processing enzymes coordinate to replicate and transcribe the viral genome, and to provide
structure-guided targets for drug discovery, with the ultimate goal of providing relief for the COVID-19
pandemic.

## Key facts

- **NIH application ID:** 10238209
- **Project number:** 1R01AI161278-01
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** ELIZABETH A CAMPBELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $638,072
- **Award type:** 1
- **Project period:** 2021-08-06 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10238209

## Citation

> US National Institutes of Health, RePORTER application 10238209, Structure, function, and inhibition of the SARS-CoV-2 replication-transcription complex (1R01AI161278-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10238209. Licensed CC0.

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