Phosphorylation of the polyomavirus major capsid protein VP1 plays a role in virus assembly and may function in virus-cell recognition. Previous mapping of the in vivo phosphorylation sites on VP1 identified phosphorylation of threonine residues Thr-63 and Thr-156 (Li, M., and Garcea, R. L.(1994) J. Virol. 68, 320-327). Phosphoserine was detected in a tryptic phosphopeptide encompassing residues 58-78. Because of consensus casein kinase II (CK II) sites in this peptide, we examined the in vitro phosphorylation of the purified recombinant VP1 protein by CK II. CK II phosphorylated VP1 on serine, and the resulting tryptic phosphopeptide eluted in a 30-31 min high performance liquid chromatography fraction corresponding to residues 58-78. The VP1 tryptic phosphopeptide also co-migrated in two-dimensional peptide analysis with one of the tryptic peptides obtained from VP1 isolated after in vivoP labeling of virus-infected cells. A site-directed mutant VP1 protein, Ser-66 to Ala, was phosphorylated poorly by CK II in vitro. As determined by electron microscopy, all of the mutant proteins were isolated in pentameric form similar to the wild-type protein, although the Ala-66 pentamers had a tendency to self-assemble in vitro into tubular as well as capsid-like structures. These findings identify Ser-66 as a site of VP1 phosphorylation in vitro, and suggest that VP1 may serve as a substrate for CK II in vivo.

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