Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template

Abstract

The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N⁴-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5'-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand.

Keywords: Covid-19; RNA-dependent RNA polymerase; SARS-CoV-2; antiviral agent; coronavirus; drug development; mutagen; nucleoside analogue.

Figure 1

Efficiency of NHC-TP incorporation.A, migration pattern of the products of RNA synthesis catalyzed by SARS-CoV-2 RNA-dependent RNA polymerase along the RNA primer/templates as shown above the panels. The sequences support incorporation of NHC-monophosphate as either a C-, U-, A-, or G-analogue at position 6. G, U, or C indicates incorporation of [α-³²P]G-, [α-³²P]U-, or [α-³²P]CTP at position 5 (red). N indicates incorporation of NHC-monophosphate. Asterisk indicates terminal transferase activity. A 5′-³²P-labeled 4-nt primer (4) serves as a size marker (m). B, graphical representation of the data shown in A. Fitting the data points to Michaelis–Menten function and the calculation of the selectivity values (Experimental procedures). Sel., selectivity for a nucleotide substrate analogue is calculated as the ratio of the V_max/K_m values for NTP over NTP analogue. Error bars illustrate standard deviation of the data. ±, standard error of the fit. All reported values have been calculated on the basis of an 8-data point experiment repeated at least three times. NHC-TP, β-D-N⁴-hydroxycytidine 5’-triphosphate.

Figure 2

SARS-CoV-2 RNA-dependent RNA polymerase–catalyzed RNA synthesis following incorporation of NHC-monophosphate. Migration pattern of the products of RNA synthesis catalyzed by SARS-CoV-2 RdRp complex along the RNA primer/template as shown at the top of the panel. RNA primer/template supports a single incorporation event of CMP or NHC-monophosphate as a C-analogue at position 6. G indicates incorporation of [α-³²P]-GTP at position 5. A 5′-³²P-labeled 4-nt primer (4) serves as a size marker (m). Asterisk indicates products of the terminal transferase activity. NHC-TP, β-D-N⁴-hydroxycytidine 5’-triphosphate.

Figure 3

RNA synthesis with NHC-MP in the template strand. Migration pattern of the reaction products catalyzed by SARS-CoV-2 RNA-dependent RNA polymerase. The template contains an embedded NHC-MP at position 11 (Template “N”) or CMP (Template “C”). Reactions with [α-³²P]-CTP as the only NTP are indicated by “0” in red. Figure notations are as in Figure 1. Asterisks indicate products of GTP misincorporation opposite U in the template. NHC-MP, β-D-N⁴-hydroxycytidine 5’-monophosphate.

Figure 4

Mechanism of template-dependent inhibition of SARS-CoV-2 RNA-dependent RNA polymerase complex. Figure notations are as in Figure 2. Signal accumulation at position 11 illustrates inhibition of nucleotide incorporation right after template-embedded β-D-N⁴-hydroxycytidine 5’-monophosphate, which can be overcome with increasing concentrations of NTP.

Figure 5

Mutagenesis model of NHC against SARS-CoV-2.A, schematic representation of SARS-CoV-2 RNA-dependent RNA polymerase (oval)-mediated nucleotide incorporation into RNA primer (gray circles)/template (white circles). Plus and minus signs indicate RNA sense. Letters A, C, G, and U refer to natural nucleotide bases. Letter M refers to molnupiravir. Three small circles refer to a triphosphate moiety of the NTP. B, alternative base pairing of NHC base moiety is supported by its tautomerization. The N-hydroxylamine form is dominant when NHC-TP is the substrate, whereas both the N-hydroxylamine and the oxime form are available when NHC-MP is embedded in the template. C, mechanism of viral inhibition and mutagenesis by template-embedded NHC-MP. Blue circles illustrate NTP incorporation past NHC-MP in the template. D, summary of NHC-mediated inhibitory and mutagenic effects on viral replication. MP, monophosphate; NHC, β-D-N⁴-hydroxycytidine; TP, 5’-triphosphate.