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

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 u...

Full description

Saved in:
Bibliographic Details
Published in:Communications chemistry 2021-03, Vol.4 (1), p.41
Main Authors: Conti, Brian J, Leicht, Andrew S, Kirchdoerfer, Robert N, Sussman, Michael R
Format: Article
Language:English
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:2399-3669
DOI:10.1038/s42004-021-00476-4