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Structural basis for substrate selection by the SARS-CoV-2 replicase

View ORCID ProfileBrandon F. Malone, View ORCID ProfileJason K. Perry, View ORCID ProfilePaul Dominic B. Olinares, View ORCID ProfileJames Chen, View ORCID ProfileTodd K. Appelby, View ORCID ProfileJoy Y. Feng, View ORCID ProfileJohn P. Bilello, Honkit Ng, Johanna Sotiris, Mark Ebrahim, View ORCID ProfileEugene Y.D. Chua, View ORCID ProfileJoshua H. Mendez, View ORCID ProfileEd T. Eng, View ORCID ProfileRobert Landick, View ORCID ProfileBrian T. Chait, View ORCID ProfileElizabeth A. Campbell, View ORCID ProfileSeth A. Darst
doi: https://doi.org/10.1101/2022.05.20.492815
Brandon F. Malone
1Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065 USA
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Jason K. Perry
2Gilead Sciences, Inc., Foster City, CA 94404 USA
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Paul Dominic B. Olinares
3Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 USA
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James Chen
1Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065 USA
7Department of Cell Biology, New York University School of Medicine, New York, NY 10016 USA
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Todd K. Appelby
2Gilead Sciences, Inc., Foster City, CA 94404 USA
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Joy Y. Feng
2Gilead Sciences, Inc., Foster City, CA 94404 USA
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John P. Bilello
2Gilead Sciences, Inc., Foster City, CA 94404 USA
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Honkit Ng
4The Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center, The Rockefeller University, New York, NY 10065, USA
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Johanna Sotiris
4The Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center, The Rockefeller University, New York, NY 10065, USA
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Mark Ebrahim
4The Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center, The Rockefeller University, New York, NY 10065, USA
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Eugene Y.D. Chua
5National Center for Cryo-EM Access and Training, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027 USA
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Joshua H. Mendez
5National Center for Cryo-EM Access and Training, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027 USA
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Ed T. Eng
5National Center for Cryo-EM Access and Training, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027 USA
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Robert Landick
6Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
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Brian T. Chait
3Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 USA
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Elizabeth A. Campbell
1Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065 USA
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  • For correspondence: darst@rockefeller.edu campbee@rockefeller.edu
Seth A. Darst
1Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065 USA
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  • ORCID record for Seth A. Darst
  • For correspondence: darst@rockefeller.edu campbee@rockefeller.edu
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Abstract

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP3,4. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5’ RNA cap6.

Competing Interest Statement

E.A.C. and S.A.D. received funding from Gilead Sciences, Inc. in support of this study. J.K.P., T.K.A., J.Y.F., and J.P.B. are Gilead employees.

Footnotes

  • ↵9 Lead contact: darst{at}rockefeller.edu

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Structural basis for substrate selection by the SARS-CoV-2 replicase
Brandon F. Malone, Jason K. Perry, Paul Dominic B. Olinares, James Chen, Todd K. Appelby, Joy Y. Feng, John P. Bilello, Honkit Ng, Johanna Sotiris, Mark Ebrahim, Eugene Y.D. Chua, Joshua H. Mendez, Ed T. Eng, Robert Landick, Brian T. Chait, Elizabeth A. Campbell, Seth A. Darst
bioRxiv 2022.05.20.492815; doi: https://doi.org/10.1101/2022.05.20.492815
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Structural basis for substrate selection by the SARS-CoV-2 replicase
Brandon F. Malone, Jason K. Perry, Paul Dominic B. Olinares, James Chen, Todd K. Appelby, Joy Y. Feng, John P. Bilello, Honkit Ng, Johanna Sotiris, Mark Ebrahim, Eugene Y.D. Chua, Joshua H. Mendez, Ed T. Eng, Robert Landick, Brian T. Chait, Elizabeth A. Campbell, Seth A. Darst
bioRxiv 2022.05.20.492815; doi: https://doi.org/10.1101/2022.05.20.492815

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