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

doi:10.1016/j.molcel.2021.10.003

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

Medicinal Chemistry

Research output: Contribution to journal › Article › peer-review

7 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 language	English (US)
Pages (from-to)	4467-4480.e7
Journal	Molecular Cell
Volume	81
Issue number	21
DOIs	https://doi.org/10.1016/j.molcel.2021.10.003
State	Published - 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

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Janissen, R., Woodman, A., Shengjuler, D., Vallet, T., Lee, K. M., Kuijpers, L., Moustafa, I. M., Fitzgerald, F., Huang, P. N., Perkins, A. L., Harki, D. A., Arnold, J. J., Solano, B., Shih, S. R., Vignuzzi, M., Cameron, C. E., & Dekker, N. H. (2021). Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses. Molecular Cell, 81(21), 4467-4480.e7. https://doi.org/10.1016/j.molcel.2021.10.003

Janissen, R, Woodman, A, Shengjuler, D, Vallet, T, Lee, KM, Kuijpers, L, Moustafa, IM, Fitzgerald, F, Huang, PN, Perkins, AL , Harki, DA, Arnold, JJ, Solano, B, Shih, SR, Vignuzzi, M, Cameron, CE & Dekker, NH 2021, 'Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses', Molecular Cell, vol. 81, no. 21, pp. 4467-4480.e7. https://doi.org/10.1016/j.molcel.2021.10.003

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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",

author = "Richard Janissen and Andrew Woodman and Djoshkun Shengjuler and Thomas Vallet and Lee, {Kuo Ming} and Louis Kuijpers and Moustafa, {Ibrahim M.} and Fiona Fitzgerald and Huang, {Peng Nien} and Perkins, {Angela L.} and Harki, {Daniel A.} and Arnold, {Jamie J.} and Bel{\'e}n Solano and Shih, {Shin Ru} and Marco Vignuzzi and Cameron, {Craig E.} and Dekker, {Nynke H.}",

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: {\textcopyright} 2021 Elsevier Inc.",

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AU - Woodman, Andrew

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AU - Vallet, Thomas

AU - Lee, Kuo Ming

AU - Kuijpers, Louis

AU - Moustafa, Ibrahim M.

AU - Fitzgerald, Fiona

AU - Huang, Peng Nien

AU - Perkins, Angela L.

AU - Harki, Daniel A.

AU - Arnold, Jamie J.

AU - Solano, Belén

AU - Shih, Shin Ru

AU - Vignuzzi, Marco

AU - Cameron, Craig E.

AU - Dekker, Nynke H.

N1 - 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.

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N2 - 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.

AB - 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.

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

Abstract

Bibliographical note

Keywords

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