511. Treatment with Molnupiravir in the MOVe-In and MOVe-Out Clinical Trials Results in an Increase in Transition Mutations Across the SARS-CoV-2 Genome

Background Molnupiravir (MOV), (MK-4482, EIDD-2801) is being clinically developed for the treatment of COVID-19 disease caused by SARS-CoV-2. MOV is the orally administered 5′-isobutyrate prodrug of the active, antiviral ribonucleoside analogue, N-hydroxycytidine (NHC, EIDD-1931) which inhibits viral replication by induction of mutations in the viral genome, leading to viral error catastrophe. In 2 clinical studies, hospitalized (MOVe-In) and non-hospitalized (MOVe-Out) participants were treated for 5 days with MOV and followed up to Day 29. Viral RNA isolated from nasal swab samples were sequenced to determine the rate, distribution and type of viral mutations observed after MOV treatment. Methods RNA isolated from nasopharangeal swab samples collected during study conduct was quantified by RT-PCR. Samples containing >22,000 copies/mL of RNA underwent complete genome NGS using the Ion AmpliSeq SARS-CoV-2 research panel and Ion Torrent sequencing. Mutation rates were calculated by determining the number of nucleotide changes observed across the entire genome at Day 3 and/or Day 5 compared to baseline. Results Combined data from both studies showed an increase of ~2-4 fold in the viral mutation rate post-baseline in MOV treated compared with placebo. Mutations were distributed across the entire genome with only a minority being observed in more than one sample. The most frequent mutations were transitions of C to U observed in the highest MOV dose group (800 mg/BID). Conclusion Consistent with the proposed mechanism of action of MOV, an increase in the rate of transition mutations in the virus was observed in post-baseline nasal swab samples from participants treated with MOV compared with placebo. Disclosures Julie Strizki, PhD, Merck & Co., Inc. (Employee, Shareholder) Jay Grobler, PhD, Merck & Co., Inc. (Employee, Shareholder) Ying Zhang, PhD, Merck & Co., Inc. (Employee, Shareholder) Jiejun Du, PhD, Merck & Co., Inc. (Employee, Shareholder) Shunbing Zhao, PhD, Merck & Co., Inc. (Employee, Shareholder) Diane Levitan, PhD, Merck & Co., Inc. (Employee, Shareholder) Alex Therien, PhD, Merck & Co., Inc. (Employee, Shareholder) Joan R. Butterton, MD, Merck Sharp & Dohme Corp. (Employee, Shareholder) Nicholas Murgolo, PhD, Merck & Co., Inc. (Employee, Shareholder)

An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

ABSTRACT Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template, whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation, while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudorevertants (L420V and L420I) were selected from all independent lineages of L420A, G64Fix L420A, and G64S L420A parental viruses. Ribavirin resistance G64S mutations were selected in two independent lineages, and novel ribavirin resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, and T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L, M392V, K375R, and R376K). L420A revertants and pseudorevertants (L420V and L420I) restored efficient viral RNA recombination, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L, and M392V) and primer grip mutations (K375R and R376K) exhibited divergent RNA recombination, ribavirin sensitivity, and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375, and R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms. IMPORTANCE Picornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses, and counteracts error catastrophe. Can viruses distinguish between homologous and nonhomologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and nonhomologous RNA templates during sexual RNA replication.

An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

ABSTRACTPicornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudo-revertants (L420V, L420I) were selected from all independent lineages of L420A, G64Fix L420A and G64S L420A parental viruses. Ribavirin-resistant G64S mutations were selected in two independent lineages and novel ribavirin-resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L & M392V and K375R & R376K). L420A revertants and pseudorevertants (L420V, L420I) restored efficient sexual RNA replication mechanisms, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L & M392V) and primer grip mutations (K375R & R376K) exhibited divergent RNA recombination, ribavirin sensitivity and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375 & R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.IMPORTANCEPicornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses and counteracts error catastrophe. Can viruses distinguish between homologous and non-homologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and non-homologous RNA templates during sexual RNA replication.

Survival of Self-Replicating Molecules under Transient Compartmentalization with Natural Selection

The problem of the emergence and survival of self-replicating molecules in origin-of-life scenarios is plagued by the error catastrophe, which is usually escaped by considering effects of compartmentalization, as in the stochastic corrector model. By addressing the problem in a simple system composed of a self-replicating molecule (a replicase) and a parasite molecule that needs the replicase for copying itself, we show that transient (rather than permanent) compartmentalization is sufficient to the task. We also exhibit a regime in which the concentrations of the two kinds of molecules undergo sustained oscillations. Our model should be relevant not only for origin-of-life scenarios but also for describing directed evolution experiments, which increasingly rely on transient compartmentalization with pooling and natural selection.

Survival of self-replicating molecules under transient compartmentalization with natural selection

The problem of the emergence and survival of self-replicating molecules in origin-of-life scenarios is plagued by the error catastrophe, which is usually escaped by considering effects of compartmentalization, as in the stochastic corrector model. By addressing the problem in a simple system composed of a self-replicating molecule (a replicase) and a parasite molecule that needs the replicase for copying itself, we show that transient (rather than permanent) compartmentalization is sufficient to the task. We also exhibit a regime in which the concentrations of the two kinds of molecules undergo sustained oscillations. Our model should be relevant not only for origin-of-life scenarios but also for describing directed evolution experiments, which increasingly rely on transient compartmentalization with pooling and natural selection.

Picornavirus RNA Recombination Counteracts Error Catastrophe

ABSTRACTTemplate-dependent RNA replication mechanisms render picornaviruses susceptible to error catastrophe, an overwhelming accumulation of mutations incompatible with viability. Viral RNA recombination, in theory, provides a mechanism for viruses to counteract error catastrophe. We tested this theory by exploiting well-defined mutations in the poliovirus RNA-dependent RNA polymerase (RDRP), namely, a G64S mutation and an L420A mutation. Our data reveal two distinct mechanisms by which picornaviral RDRPs influence error catastrophe: fidelity of RNA synthesis and RNA recombination. A G64S mutation increased the fidelity of the viral polymerase and rendered the virus resistant to ribavirin-induced error catastrophe, but only when RNA recombination was at wild-type levels. An L420A mutation in the viral polymerase inhibited RNA recombination and exacerbated ribavirin-induced error catastrophe. Furthermore, when RNA recombination was substantially reduced by an L420A mutation, a high-fidelity G64S polymerase failed to make the virus resistant to ribavirin. These data indicate that viral RNA recombination is required for poliovirus to evade ribavirin-induced error catastrophe. The conserved nature of L420 within RDRPs suggests that RNA recombination is a common mechanism for picornaviruses to counteract and avoid error catastrophe.IMPORTANCEPositive-strand RNA viruses produce vast amounts of progeny in very short periods of time via template-dependent RNA replication mechanisms. Template-dependent RNA replication, while efficient, can be disadvantageous due to error-prone viral polymerases. The accumulation of mutations in viral RNA genomes leads to error catastrophe. In this study, we substantiate long-held theories regarding the advantages and disadvantages of asexual and sexual replication strategies among RNA viruses. In particular, we show that picornavirus RNA recombination counteracts the negative consequences of asexual template-dependent RNA replication mechanisms, namely, error catastrophe.

Daniel McGillivray Brown. 3 February 1923—24 April 2012

Dan Brown was a nucleic acids chemist of the highest order, beginning with pioneering work under Lord Alexander Todd in the 1950s at University of Cambridge on chemical methods for synthesis of nucleosides and nucleotides. This work helped to confirm the furanose chemical structure of the sugar in nucleosides as well as the 3′-5′ phosphodiester linkage in DNA and RNA, perhaps the most well thought of achievement of his career. Later, as a chemistry department lecturer, he established the chemical structures of glycerol monophosphoinositides as well as triphosphoinositides. Turning back to the nucleic acids in 1961, he became fascinated by the effect of mutagens on DNA. He elucidated the mechanism for the reaction of hydroxylamine on cytidine to form an initial ‘bis-adduct’ and thereafter N 6 -hydroxycytidine. Moving in 1982 to the MRC Laboratory of Molecular Biology, he developed a method to prepare single-stranded DNA probes for detection of RNA sequences and in addition worked on a novel automated device for oligonucleotide synthesis. Reverting to his interest in mutagens, he then designed and synthesized hydrogen bonding degenerate bases and developed novel P and K modified pyrimidine and purine bases respectively as transition mutagens. Finally, he synthesized the base analogue 5-nitroindole as a potential universal base, which became useful in cycle DNA sequencing, and in addition developed the concept of ‘error catastrophe’ for the ribonucleoside of the P base as an antiviral agent. The P, K and 5-nitroindole bases became the most valued chemical entities of his career to molecular biologists. His legacy to the nucleic acids includes both his significant contributions to studies of the chemical nature of DNA and RNA and their constituents as well as a variety of enabling nucleic acids chemistry methods and mechanisms of DNA mutagenicity.