Mass Spectrometric Based Detection of Protein Nucleotidylation in the RNA Polymerase of SARS-CoV-2

Overview

Journal Commun Chem

Publisher Springer Nature

Specialty Chemistry

Date 2023 Jan 25

PMID 36697572

Authors

Brian J Conti

Andrew S Leicht

Robert N Kirchdoerfer

Michael R Sussman

Affiliations

Soon will be listed here.

Abstract

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

References

Polack F, Thomas S, Kitchin N, Absalon J, Gurtman A, Lockhart S . Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020; 383(27):2603-2615. PMC: 7745181. DOI: 10.1056/NEJMoa2034577. View

Arts E, Hazuda D . HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect Med. 2012; 2(4):a007161. PMC: 3312400. DOI: 10.1101/cshperspect.a007161. View

Bekker L, Tatoud R, Dabis F, Feinberg M, Kaleebu P, Marovich M . The complex challenges of HIV vaccine development require renewed and expanded global commitment. Lancet. 2019; 395(10221):384-388. DOI: 10.1016/S0140-6736(19)32682-0. View

Falade-Nwulia O, Suarez-Cuervo C, Nelson D, Fried M, Segal J, Sulkowski M . Oral Direct-Acting Agent Therapy for Hepatitis C Virus Infection: A Systematic Review. Ann Intern Med. 2017; 166(9):637-648. PMC: 5486987. DOI: 10.7326/M16-2575. View

Scott N, Wilson D, Thompson A, Barnes E, El-Sayed M, Benzaken A . The case for a universal hepatitis C vaccine to achieve hepatitis C elimination. BMC Med. 2019; 17(1):175. PMC: 6749704. DOI: 10.1186/s12916-019-1411-9. View

Gordon C, Tchesnokov E, Woolner E, Perry J, Feng J, Porter D . Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J Biol Chem. 2020; 295(20):6785-6797. PMC: 7242698. DOI: 10.1074/jbc.RA120.013679. View

Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L . Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020; 368(6492):779-782. PMC: 7164392. DOI: 10.1126/science.abb7498. View

Kirchdoerfer R, Ward A . Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat Commun. 2019; 10(1):2342. PMC: 6538669. DOI: 10.1038/s41467-019-10280-3. View

Lehmann K, Gulyaeva A, Zevenhoven-Dobbe J, Janssen G, Ruben M, Overkleeft H . Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses. Nucleic Acids Res. 2015; 43(17):8416-34. PMC: 4787807. DOI: 10.1093/nar/gkv838. View

10.

Yang G, Zhou R, Guo X, Yan C, Lei J, Shi Y . Structural basis of γ-secretase inhibition and modulation by small molecule drugs. Cell. 2020; 184(2):521-533.e14. DOI: 10.1016/j.cell.2020.11.049. View

11.

Fresco L, Buratowski S . Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases. Proc Natl Acad Sci U S A. 1994; 91(14):6624-8. PMC: 44255. DOI: 10.1073/pnas.91.14.6624. View

12.

Ramanathan A, Robb G, Chan S . mRNA capping: biological functions and applications. Nucleic Acids Res. 2016; 44(16):7511-26. PMC: 5027499. DOI: 10.1093/nar/gkw551. View

13.

Toyama R, Mizumoto K, Nakahara Y, Tatsuno T, Kaziro Y . Mechanism of the mRNA guanylyltransferase reaction: isolation of N epsilon-phospholysine and GMP (5' leads to N epsilon) lysine from the guanylyl-enzyme intermediate. EMBO J. 1983; 2(12):2195-201. PMC: 555434. DOI: 10.1002/j.1460-2075.1983.tb01723.x. View

14.

Subissi L, Posthuma C, Collet A, Zevenhoven-Dobbe J, Gorbalenya A, Decroly E . One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci U S A. 2014; 111(37):E3900-9. PMC: 4169972. DOI: 10.1073/pnas.1323705111. View

15.

Chen J, Xu X, Tao H, Li Y, Nan H, Wang Y . Structural Analysis of Porcine Reproductive and Respiratory Syndrome Virus Non-structural Protein 7α (NSP7α) and Identification of Its Interaction with NSP9. Front Microbiol. 2017; 8:853. PMC: 5425468. DOI: 10.3389/fmicb.2017.00853. View

16.

Manolaridis I, Gaudin C, Posthuma C, Zevenhoven-Dobbe J, Imbert I, Canard B . Structure and genetic analysis of the arterivirus nonstructural protein 7alpha. J Virol. 2011; 85(14):7449-53. PMC: 3126611. DOI: 10.1128/JVI.00255-11. View

17.

Duclos B, Marcandier S, Cozzone A . Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol. 1991; 201:10-21. DOI: 10.1016/0076-6879(91)01004-l. View

18.

Chen J, Malone B, Llewellyn E, Grasso M, Shelton P, Olinares P . Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex. Cell. 2020; 182(6):1560-1573.e13. PMC: 7386476. DOI: 10.1016/j.cell.2020.07.033. View

19.

Riley N, Coon J . The Role of Electron Transfer Dissociation in Modern Proteomics. Anal Chem. 2017; 90(1):40-64. PMC: 5750139. DOI: 10.1021/acs.analchem.7b04810. View

20.

Penkert M, Hauser A, Harmel R, Fiedler D, Hackenberger C, Krause E . Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations. J Am Soc Mass Spectrom. 2019; 30(9):1578-1585. DOI: 10.1007/s13361-019-02240-4. View