Copy-back RNA synthesis by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir

Rakib A, Das A, Bera SC, Cnossen J, Robles-Sikisaka R, Diefenbacher MV, America PP, Klein M, Anderson TK, Marecki JC, Wang B, Bogers E, Feng JY, Bilello JP, Papini FS, Smitskamp Q, Sheahan TP, Arnold JJ, Routh A, Artsimovitch I, Kirchdoerfer RN, Cameron CE, Raney KD, Dulin D (2026)

Publication Type: Journal article

Publication year: 2026

Journal

Science Advances American Association for the Advancement of Science

Book Volume: 12

Journal Issue: 25

DOI: 10.1126/sciadv.adz9231

Abstract

Polymerase template switching is an essential mechanism in coronaviruses (CoVs) enabling both subgenomic RNA synthesis and increasing genomic diversity via recombination. Despite its importance, the CoV polymerase template-switching molecular mechanism remains unclear. Using magnetic tweezers, we show that the CoV nonstructural protein (nsp) 13-helicase drives intramolecular polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase adenosine triphosphatase activity and a duplex RNA downstream of the CoV polymerase. Remdesivir and molnupiravir are antiviral nucleotide analogs reported to stall the viral polymerase and induce mutations in genome, respectively. Unexpectedly, we show that their incorporation in the nascent strand increases copy-back RNA synthesis in vitro and decreases recombination events in infected cells. We propose a mechanism of action where these analogs' incorporation traps replication complex in a recombination intermediate, preventing viral RNA utilization. Our study highlights the importance of investigating nucleotide analog mechanisms in replication complexes beyond the polymerase.

Authors with CRIS profile

Subhas Chandra Bera Interdisziplinäres Zentrum für Klinische Forschung IZKF Nachwuchsgruppe 2 Prof. Dr. Dulin

Involved external institutions

Vrije Universiteit Amsterdam (VU) / University Amsterdam NL Netherlands (NL) Scripps Research Institute (TSRI) US United States (USA) (US) University of North Carolina at Chapel Hill US United States (USA) (US) University of Wisconsin - Madison US United States (USA) (US) University of Arkansas for Medical Sciences (UAMS) US United States (USA) (US) Emory University US United States (USA) (US) Gilead Sciences, Inc. US United States (USA) (US) Ohio State University US United States (USA) (US)

How to cite

APA:

Rakib, A., Das, A., Bera, S.C., Cnossen, J., Robles-Sikisaka, R., Diefenbacher, M.V.,... Dulin, D. (2026). Copy-back RNA synthesis by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir. Science Advances, 12(25). https://doi.org/10.1126/sciadv.adz9231

MLA:

Rakib, Asif, et al. "Copy-back RNA synthesis by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir." Science Advances 12.25 (2026).

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