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The MNV-1 protease-polymerase precursor cleaves a novel site in the NS1-2 protein

Vernon K Ward1,2, Geena M McKenzie-Goldsmith1, Matt J Edwards1

  • 1Department of Microbiology and Immunology, Faculty of Biomedical Sciences, University of Otago, PO Box 56, Dunedin 9054, New Zealand.

The Journal of General Virology
|April 29, 2026
PubMed

Insights

Murine norovirus (MNV) non-structural protein 1-2 (NS1-2) cleavage by the protease-polymerase (ProPol) precursor is crucial for viral replication. This specific cleavage site is essential for efficient viral protein expression and high viral yields.

Area of Science:

  • Virology
  • Molecular Biology
  • Structural Biology

Background:

  • Human noroviruses (HuNVs) cause significant gastroenteritis globally, but murine norovirus (MNV) is a key model for studying norovirus replication due to challenges with HuNV cell culture.
  • Norovirus genomes encode a non-structural (NS) polyprotein processed by viral proteases into functional proteins during replication.
  • Understanding the precise mechanisms of norovirus polyprotein processing is vital for developing antiviral strategies.

Purpose of the Study:

  • To identify and characterize a specific cleavage site within the MNV-1 NS1-2 protein.
  • To investigate the role of this cleavage site and its processing by the protease-polymerase (ProPol) precursor in viral replication.
  • To determine the impact of mutations at this cleavage site on viral protein expression and yield.

Main Methods:

  • Sequence analysis to predict a putative viral protease cleavage site (E66/G67) in MNV-1 NS1-2.
  • In vitro cleavage assays using the ProPol precursor and mature protease.
  • Mass spectrometry (MS) to confirm the cleavage site.
  • Site-directed mutagenesis to alter the cleavage site motif and assess viral replication in cell culture models (Huh7CD300lf and BV2 cells).

Main Results:

  • A putative cleavage site at E66/G67 in MNV-1 NS1-2 was identified and confirmed by MS, showing cleavage by the ProPol precursor but not the mature protease.
  • This cleavage site is conserved across MNV strains, with some exhibiting a predicted caspase cleavage site.
  • Mutating the ProPol cleavage site to a caspase motif reduced viral protein expression and replication.
  • Ablation of the E66/G67 cleavage site (E66A mutation) resulted in a 100-fold reduction in viral yield in BV-2 cells.

Conclusions:

  • The protease function of the MNV-1 ProPol precursor plays a critical, distinct role in viral replication compared to the mature protease.
  • Cleavage of NS1-2 by ProPol at the E66/G67 site is essential for efficient MNV-1 replication.
  • This finding highlights a key step in norovirus replication mediated by the ProPol precursor.
Keywords:
NS1-2murine noroviruspolyprotein processingprecursor proteinproteaseprotease–polymerase (ProPol)

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