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Bovine coronavirus nonstructural protein ns2 is a phosphoprotein

G J Cox1, M D Parker, L A Babiuk

  • 1Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.

Virology
|November 1, 1991
PubMed

Insights

Researchers characterized the bovine coronavirus (BCV) ns2 protein, finding it to be a stable, phosphorylated nonstructural protein that accumulates in infected cells. This study marks the first identification of a phosphorylated nonstructural protein in coronaviruses.

Area of Science:

  • Virology
  • Molecular Biology
  • Protein Biochemistry

Background:

  • Bovine coronavirus (BCV) is a significant pathogen in cattle.
  • The nonstructural protein 2 (ns2) of BCV plays a role in viral replication, but its nature remains poorly understood.
  • Understanding viral protein functions is crucial for developing effective antiviral strategies.

Purpose of the Study:

  • To investigate the biochemical properties and cellular localization of the BCV ns2 protein.
  • To determine if the ns2 protein is modified post-translationally, specifically through phosphorylation.
  • To establish the stability and accumulation patterns of ns2 during BCV infection.

Main Methods:

  • Cloning and expression of the BCV ns2 gene as a beta-galactosidase fusion protein.
  • Generation of specific antiserum against the ns2 protein.
  • Indirect immunofluorescence microscopy to detect ns2 in infected cells.
  • In vitro synthesis and immunoprecipitation to confirm protein size and specificity.
  • Pulse-chase experiments to assess protein stability and accumulation.
  • [32P] orthophosphate labeling and phosphoamino acid analysis to identify post-translational modifications.

Main Results:

  • The ns2 gene was successfully cloned and expressed, producing a detectable protein.
  • Antiserum specifically recognized the ns2 protein in infected cells and in vitro synthesized products (~32 kDa).
  • ns2 protein synthesis mirrored that of structural viral proteins and accumulated in infected cells.
  • Pulse-chase experiments confirmed ns2 protein stability.
  • Metabolic labeling revealed ns2 is a phosphoprotein, with serine and threonine identified as phosphorylation sites.

Conclusions:

  • The bovine coronavirus ns2 protein is a stable, phosphorylated nonstructural protein.
  • ns2 accumulates in infected cells, suggesting a significant role in the viral life cycle.
  • This study provides the first evidence of a phosphorylated nonstructural protein in coronavirus-infected cells, opening new avenues for research into coronavirus pathogenesis and replication.

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