β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells

Abstract

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: NHC; SARS-CoV-2; molnupiravir; mutagenicity.

Figure 1.

Antiviral and mutagenic effect of rNHC, RBV, and FAV on SARS-CoV-2 in the A549-hACE2 cell model. A, Antiviral effect of rNHC, RBV, and FAV on SARS-CoV-2. Significance compared to DMSO control (* P < .05) was determined using a 2-stage linear t test with step-up procedure of Benjamini, Krieger, and Yekutieli through the GraphPad Prism version 8.3.0 built-in function. B, Total nucleotide substitution rate in the SARS-CoV-2 viral genome within the nsp12 coding region. C, Cytidine to uridine substitution rate of the SARS-CoV-2 viral genome within the nsp12 region. D, Correlation of the cytidine to uridine mutation level within the nsp12 region to the loss of infectivity relative to the DMSO control. Linear regression lines are shown as dashed lines for each drug group, and the shadows indicate the confidence intervals of the regressions. Abbreviations: DMSO, dimethyl sulfoxide; FAV, favipiravir; PFU, plaque-forming unit; RBV, ribavirin; rNHC, β-d-N⁴-hydroxycytidine; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

HPRT assay to detect genotoxicity of rNHC, RBV, FAV, AZT, 3TC, and TDF in CHO-K1 cells. A, 6-thioguanine-resistant colony counts in 2 separate HPRT mutagenesis experiments. In the HPRT experiment 2, an additional round of initial cleansing for spontaneous HPRT mutations was conducted to limit background mutations. Each compound/dose group had 3 replicates. Average numbers of colonies are shown on the top of each bar. Significance compared to vehicle control (* P = .01–.05, ** P = .001–.01, **** P <.0001) was determined using the unpaired t test calculated using the GraphPad Prism version 8.3.0 built-in function. B, Highlighter plot of the HPRT mutation colony sequencing from the second experiment. 6-TG–resistant colonies were scraped from the cell culture dish and transferred into 24-well tissue culture plate wells in complete growth medium with 30 µM 6-TG for an additional 4 days to expand the cells. Cells in each well were collected and total RNA was extracted. We amplified HPRT mRNA using 1-step RT-PCR, and sequenced the PCR products with Sanger sequencing. The total sequenced region was 883 bases of the total 1179-base HPRT mRNA (XM_007643626.2), with regions on each end of the mRNA not covered by sequencing. Each colony sequence was compared with the reference mRNA sequence. A total of 42 colonies were sequenced and 32 of them had missense substitutions or frame shifts from deletions within the region sequenced. Most of the mutations were different, while a few colonies contained the identical mutation, consistent with expansion after a mutation early in the treatment cycle. Abbreviations: 3TC, lamivudine; AZT, zidovudine; FAV, favipiravir; RBV, ribavirin; rNHC, β-d-N⁴-hydroxycytidine; REF, reference; RT-PCR, reverse transcription polymerase chain reaction; TDF, tenofovir.