panoramaProteomicsLeandro NevesMurine NorovirusMus Musculushttps://panoramaweb.org/LN_VPg_nucleotidylation_2026.urlleandroxneves@gmail.comCentre for Proteome Research, Dept. Biochemistry & Systems Biology, University of Liverpool, Liverpool, UKPanoramaPublicNorovirus is a leading cause of global acute gastroenteritis, yet the mechanisms underlying its replication remain incompletely understood. As a positive-sense single-stranded RNA (+ ssRNA) virus, norovirus utilizes a nucleotidylated viral protein (VPg) as a primer for synthesis of its genomic and subgenomic mRNAs. In this study, we report the efficient solid-phase synthesis of a guanylylated (also known as GMPylated) VPg-derived peptide fragment using a novel pre-guanylylated tyrosine building block equipped with acid-sensitive protecting groups. This approach enables a streamlined, one-step deprotection and cleavage process. The resulting synthetic peptide was characterised via LC-MS/MS, establishing its gas-phase fragmentation behaviour and confirming prior observations of a diagnostic guanine nucleobase peak at 152.0572 m/z. We subsequently applied these findings to develop a parallel reaction monitoring (PRM) assay to investigate endogenous VPg guanylylation in murine microglial cells infected with murine norovirus (MNV). Our results demonstrate that while unmodified VPg is detectable from 4 hours post-infection, the guanylylated form appears predominantly during later stages (8-12 hpi). The timing of VPg guanylylation is consistent with a model where guanylylation may serve as a regulator in the switch between anti-sense and sense RNA synthesis, though at this time this hypothesis is speculative and remains to be proven. These synthetic molecular tools and mass spectrometry assays provide a robust framework for further exploring the role of nucleotidylation in viral pathogenesis and in broader cell and pathogen biology.Synthesis of a guanylylated peptide derived from norovirus VPg as a molecular tool for studying norovirus replication.van Puffelen Bob B, Neves Leandro Xavier LX, Steneker Roy R, Meeuwenoord Nico J NJ, Emmott Edward E, Filippov Dmitri V DVfalseSynthesis of guanylylated peptides derived from norovirus VPg as molecular tools for studying norovirus replication C4PR_LIVNorovirus, a member of the Caliciviridae family, is a positive-sense single-stranded RNA (+ssRNA) virus and a leading cause of acute gastroenteritis worldwide, especially affecting young children and the elderly. Due to a lack of specific treatment, norovirus infection causes 200,000 deaths each year and imposes an economic burden exceeding $60 billion. To address this challenge and develop medicines and diagnostics, further research into norovirus reproduction is needed. Norovirus uses a nucleotidylated protein, known as VPg (viral protein genome-linked), to initiate genome replication, by acting as a replication primer. The genomic RNA of norovirus is linked by a phosphodiester bond between the 5’-terminal guanosine (norovirus) and the phenolic oxygen of a tyrosine residue (Y27 for norovirus). In this work we demonstrate the synthesis of peptides derived from guanylylated norovirus VPg and their use in proteomic studies of norovirus replication.Tue Sep 08 00:00:00 GMT+01:00 2026PXD0750743572311009042614786