Abstract: The Journal of Infectious Diseases Brief Report β-d-N4-hydroxycytidine Inhibits SARSCoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells Shuntai Zhou,1, Collin S. Hill,1 Sanjay Sarkar,2 Longping V. Tse,3 Blaide M. D. Woodburn,1,4 Raymond F. Schinazi,5 Timothy P. Sheahan,3 Ralph S. Baric,3,6 Mark T. Heise,2,6 and Ronald Swanstrom1,7 Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. β-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host. Keywords. molnupiravir; mutagenicity; NHC; SARS-CoV-2. Emerging RNA viruses arising from highly heterogeneous pools of precursor strains are responsible for most recent epidemic and pandemic disease outbreaks in the late 20th and early 21st century. Broad direct-acting antiviral agents represent the most specific way of treating a viral infection, although these are often specific to a group of closely related viruses. Besides remdesivir, which blocks the replication of several coronaviruses, limited therapeutic options are available for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections [1]. β-d-N4-hydroxycytidine (NHC, or to designate Received 18 February 2021; editorial decision 1 May 2021; accepted 4 May 2021; published online May 7, 2021. Correspondence: Ronald Swanstrom, PhD, Room 22-006, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599 (risunc@med.unc. edu). The Journal of Infectious Diseases®  2021;224:415–9 © The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. DOI: 10.1093/infdis/jiab247 METHODS We measured SARS-CoV-2 antiviral activity in the presence of a panel of compounds (rNHC, RBV, and FAV) using the A549-hACE2 cell model [9, 10]. Sequence analysis to detect the mutation load was done using the previously published multiplex Primer ID approach to sequence several regions of the BRIEF REPORT • jid 2021:224 (1 August) • 415 1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, 2Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, 3Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, 4Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, 5Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, Georgia, USA, 6Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North..