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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
Summary
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.