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

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

19 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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10.1016/j.molcel.2021.10.003

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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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title = "Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses",

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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doi = "10.1016/j.molcel.2021.10.003",

language = "English (US)",

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T1 - Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses

AU - Janissen, Richard

AU - Woodman, Andrew

AU - Shengjuler, Djoshkun

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.

PY - 2021/11/4

Y1 - 2021/11/4

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.

KW - RNA-dependent RNA polymerase

KW - T-1106

KW - backtracking

KW - copy-back RNA synthesis

KW - enterovirus A71

KW - favipiravir

KW - poliovirus

KW - pyrazine-carboxamide analogue

KW - recombination

KW - template switching

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UR - https://www.scopus.com/pages/publications/85118473739#tab=citedBy

U2 - 10.1016/j.molcel.2021.10.003

DO - 10.1016/j.molcel.2021.10.003

M3 - Article

C2 - 34687604

AN - SCOPUS:85118473739

SN - 1097-2765

VL - 81

SP - 4467-4480.e7

JO - Molecular Cell

JF - Molecular Cell

IS - 21

ER -

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

Abstract

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Keywords

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