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J. Microbiol. Biotechnol. 1992; 2(2): 122-128

Published online June 28, 1992

Copyright © The Korean Society for Microbiology and Biotechnology.

Phosphorylation of the Nucleocapsid Protein of Bovine Coronavirus Expressed with a Recombinant Baculovirus Vector

Dongwan Yoo *, Graham J. Cox * and Ick-Dong Yoo 1*

Abstract

Post-translational modifications of the nucleocapsid protein of bovine coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic $[^{32}P]$orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of bovine coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by $[^{32}P]$orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of bovine coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.

Keywords: Bovine coronavirus, Phosphorylation, Nucleocapsid, Baculovirus vector, Expression, Sf9 cells