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.
Bibliographical noteFunding Information:
We acknowledge funding to S.-R.S., C.E.C., and N.H.D. from the Human Frontiers Science Program ( RPG0011/2015 ); to C.E.C. and J.J.A. from the National Institutes of Health ( R01AI45818 ); to A.W. from the American Heart Association ( 18POST33960071 ); and to S.-R.S. from the Ministry of Science and Technology, Taiwan (MOST; 107-3017-F-182-001 ) and the Research Center for Emerging Viral Infections . We thank Friso Douma and Wessel Teunisse for experimental assistance, Theo van Laar for RdRp purification and RNA construct synthesis, and Martin Depken, David Dulin, and Behrouz Eslami-Mossallam for fruitful discussions.
© 2021 Elsevier Inc.
- RNA-dependent RNA polymerase
- copy-back RNA synthesis
- enterovirus A71
- pyrazine-carboxamide analogue
- template switching
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't