Bovine coronavirus nonstructural protein ns2 is a phosphoprotein

To investigate the nature of the bovine coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a β-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [31P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in coronavirus-infected cells.