Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region

Abstract

The major virion protein of polyomavirus, VP1, consists of about six isoelectric species designated A through F. The minor species D, E, and F are phosphorylated and are thought to serve as viral receptors. We first wanted to distinguish whether all VP1 species are derived by post-translational modification from a common amino acid sequence or whether one or more of the species contain a region(s) of altered amino acid sequence resulting from alternate mRNA processing. We compared the VP1 species by detailed peptide mapping with several combinations of specific protease and radioisotopic labels. This approach enabled us to examine more than 80% of the predicted VP1 sequence, including the amino-and carboxy-termini. We found no evidence of sequence differences among any of the VP1 species. The specific incorporation of 32Pi was found to be the same for all of the phosphorylated species. Comparison of the phosphorylation sites of in vivo 32Pi-labeled D, E, and F by peptide mapping showed them to be identical. Each phosphorylated species contained a single major phosphopeptide and several minor phosphopeptides. The major phosphoamino acid, identified by acid hydrolysis, was phosphothreonine, with phosphoserine also present. By using chemical cleavage methods, we localized the major phosphorylation region to a central portion of the VP1 sequence. We discuss some features of this region and relate this information to functional implications of phosphorylation.

Differences in biological activity and structural protein VP1 phosphorylation of polyomavirus progeny resulting from infection of primary mouse kidney and primary mouse embryo cell cultures

“…Virus was labeled with 32P by the addition of serum-free, phosphate-free Eagle medium containing 60 pLCi of 32P, (carrier free; ICN) per ml to the appropriate cell cultures 18 h postinfection as described previously (2,3). CsCl gradient-purified polyomavirus virions were labeled in vitro with 1251 (Amersham Corp., Arlington Heights, Ill.) by using the chloramine-T method as described by Frost and Bourgaux (14).…”

“…One-dimensional gel electrophoresis was performed in a 15% sodium dodecyl sulfate-polyacrylamide gel system as described by Laemmli (20). Two-dimensional gel electrophoresis was performed as described previously (2,3,28,29), except that polybuffer 74 (Pharmacia, Inc., Piscataway, N.J.) (6% [vol/vol]) and polybuffer 96 (1% [vol/vol]) were used in place of ampholytes for the IEF. Proteins were visualized by Coomassie blue staining of the gels.…”

“…Each of these species has the same amino acid sequence, as demonstrated by peptide mapping (2). This charge heterogeneity is due at least in part to phosphorylation (1-3, 15, 29, 30), since three of the identified species (D, E, and F) are phosphorylated (2,3). Our laboratory is particularly interested in these three phosphorylated species since we have shown previously that these species correlate with the hemagglutination ability of the virus as well as specific cellular attachment (1,(3)(4)(5).…”

“…Charge heterogeneity of VP1 can be demonstrated by isoelectric focusing (IEF), in which this protein can be resolved into six species designated A to F (2,3,38). Each of these species has the same amino acid sequence, as demonstrated by peptide mapping (2).…”

“…Charge heterogeneity of VP1 can be demonstrated by isoelectric focusing (IEF), in which this protein can be resolved into six species designated A to F (2,3,38). Each of these species has the same amino acid sequence, as demonstrated by peptide mapping (2). This charge heterogeneity is due at least in part to phosphorylation (1-3, 15, 29, 30), since three of the identified species (D, E, and F) are phosphorylated (2,3).…”