IDENTIFICATION OF THE THREONINE PHOSPHORYLATION SITES ON THE POLYOMAVIRUS MAJOR CAPSID PROTEIN VP1 - RELATIONSHIP TO THE ACTIVITY OF MIDDLET-ANTIGEN

Phosphorylation of the polyomavirus major capsid protein VP1 nas exami ned after in vivo P-32 labeling of virus-infected cells. Two phosphory lated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry , and N-terminal sequencing. The peptides from residues 58 to 78 and r esidues 153 to 173 were phosphorylated on threonine. Site-directed mut ations were introduced at these threonine sites, and mutant viruses we re reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) re sulted in viruses defective in phosphorylation of the respective pepti des after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient i n assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation wh en grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T . L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections dem onstrated that both residues T-63 and T-156 were affected. These resul ts suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.