Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses

Mol Cell. 2021 Nov 4;81(21):4467-4480.e7. doi: 10.1016/j.molcel.2021.10.003. Epub 2021 Oct 22.

Abstract

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Keywords: RNA-dependent RNA polymerase; T-1106; backtracking; copy-back RNA synthesis; enterovirus A71; favipiravir; poliovirus; pyrazine-carboxamide analogue; recombination; template switching.