Synthesis of a guanylylated peptide derived from norovirus VPg as a molecular tool for studying norovirus replication

Norovirus 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.