An investigational study into the drug-associated mutational signature in SARS-CoV-2 viruses

Abstract

Novel emerging viruses have consistently posed risks to global public health through causing disease outbreaks, the most recent of which being COVID-19 pandemic in late 2019 caused by a novel coronavirus SARS-CoV-2. An orally available lethal mutagenic antiviral, molnupiravir, was approved in late 2021 for use as a COVID-19 antiviral. Lethal mutagenic antiviral agents often have broad-spectrum antiviral activity and can effectively reduce the viral population size by increasing replication errors within RNA viruses. However, inadequate concentrations of drug may result in sublethal mutagenesis, leading to accelerated intra-host evolution rates and the potential emergence of viruses with increased fitness from this viral population. Currently, there is a lack of clear data on the mutational burden induced by various mutagenic approved drugs on SARS-CoV-2 and the impact of the resulting drug-associated mutations on the fitness and pathogenicity of SARS-CoV-2. In this study, a 30-day passage of SARS-CoV-2 on VeroE6-TMPRSS2 in presence of various approved drugs with potential mutagenic effects was conducted. In addition to molnupiravir, other approved drugs with mutagenic potential were also tested, including orally available lethal mutagenic antivirals favipiravir and ribavirin, HIV antiretrovirals zidovudine, lamivudine, tenofovir and emtricitabine, antineoplastic drugs 5-azacytidine, 5-fluorouracil, 6-thioguanine, gemcitabine and cisplatin. Viral supernatants collected at the end of the passage study were evaluated on plaque assays for inspection of potential virological changes. Based on plaque morphologies of the passaged viral quasispecies, some drugs were selected for further sequencing studies and evaluation of in vitro and in vivo comparative pathogenicity. Next generation sequencing techniques were used to obtain the de novo mutational spectra present in different drug-treated viral quasispecies, as well as the single base substitution (SBS) mutational signatures associated with various drug treatments at the end of the passage study and the impact of various drugs on nucleotide diversity and evolution pressure. Lethal mutagenic antivirals molnupiravir, favipiravir and ribavirin were associated with elevated transition mutations in asymmetrical manner, in particular C>T substitutions, as well as A>G substitutions in molnupiravir-treated populations. Antineoplastic 5-fluorouracil also had similar increases in C>T and A>G transitions. 5-Azacytidine was associated with a significantly elevated C>G and modest increases in C>A transversions. Favipiravir and 5-azacytidine treatments induced the highest increases in nucleotide diversity, but the two viral populations were found to have evolved under different selection pressure. Comparative pathogenicity of the viruses were assessed on in vitro cell models to compare the viral replication kinetics on VeroE6-TMPRSS2 and immune gene expression profiles on Calu-3 cells. While the replication efficiency of mutated viruses showed no significant differences, their ability to stimulate proinflammatory and IFN-related immune responses varied. Notably, a proportion of viruses obtained from passaging in presence of molnupiravir formed significantly larger viral plaques compared to controls and was found to carry the envelope protein V5G mutation. This strain induced a stronger proinflammatory response coupled with a weaker IFN-related response in infected hamsters. On the other hand, viruses passaged in presence of 5-AzaC appeared to induce milder disease and cause attenuated proinflammatory responses in infected hamsters.