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

Richard Janissen, Andrew Woodman, Djoshkun Shengjuler, Thomas Vallet, Kuo Ming Lee, Louis Kuijpers, Ibrahim M. Moustafa, Fiona Fitzgerald, Peng Nien Huang, Angela L. Perkins, Daniel A. Harki, Jamie J. Arnold, Belén Solano, Shin Ru Shih, Marco Vignuzzi, Craig E. Cameron, Nynke H. Dekker

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)4467-4480.e7
JournalMolecular Cell
Volume81
Issue number21
DOIs
StatePublished - Nov 4 2021

Bibliographical note

Funding 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.

Publisher Copyright:
© 2021 Elsevier Inc.

Keywords

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

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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