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The effect of polyoma middle-sized tumor antigen (MTAg) on phosphatidylinositol metabolism has been characterized in vivo and in vitro using polyoma-transformed and polyoma-infected cells. Cells infected with transformation-competent polyoma virus exhibit increased levels of inositol phospholipids and the second messenger inositol trisphosphate. MTAg or pp60c-src immunoprecipitates from MTAg-transformed cells contain an activity that phosphorylates phosphatidylinositol and phosphatidylinositol 4-phosphate. This activity is induced in parallel with MTAg when the MTAg synthesis is regulated by hormonal or heavy metal inducers. Immunoprecipitates from one class of polyoma mutants defective in transformation have a reduced level of associated phosphatidylinositol kinase activity in vitro yet are capable of tyrosine phosphorylation on exogenous protein substrates at rates comparable to wild-type virus. Thus, for these mutants, phosphatidylinositol kinase activity is more tightly correlated with transformation than is protein kinase activity. These results suggest that alterations in phosphatidylinositol metabolism by MTAg play a role in transformation by polyoma virus.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.

We have expression-cloned in Escherichia coli the major polyoma virus capsid protein, VP1. Under the inducible control of the hybrid tac promoter, VP1 constituted between 2 and 3% of the total host cell protein. The expressed VP1 was purified to near homogeneity with initial yields to 10%. Optimal expression was temperature-dependent, and significant intracellular degradation could be demonstrated. The final product was obtained as one predominant isoelectric focusing species, without the pattern of post-translational modification seen in virus-infected eukaryotic cells. The purified VP1 from E. coli will be useful as a substrate for the purification of VP1 modification enzymes and in the study of inter-VP1 oligomerization.

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 vivo 32P 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.

The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP1 protein stimulated [3H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins.