Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Brian J Conti; Andrew S Leicht; Robert N Kirchdoerfer; Michael R Sussman

doi:10.1038/s42004-021-00476-4

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Commun Chem. 2021:4:41. doi: 10.1038/s42004-021-00476-4. Epub 2021 Mar 19.

Authors

Brian J Conti^{1

2}, Andrew S Leicht^{1

3}, Robert N Kirchdoerfer^{1

3}, Michael R Sussman^{1

2}

Affiliations

¹ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
² Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI 53706.
³ Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706.

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.

Abstract

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