Mapping of phosphorylation sites in polyomavirus large T antigen.

The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.     

Backbone assignment of the N-terminal polyomavirus large T antigen

Polyoma Large T antigen (PyLT) is a viral oncoprotein that targets cell proteins important for growth regulation. PyLT has two functional domains. Here we report ¹H, ¹⁵N, ¹³C backbone and ¹³C beta assignments of 76% of the residues of the polyomavirus large T antigen N-terminal domain (PyLTNT) that is sufficient to regulate cell phenotype. PyLTNT is substantially unfolded even in regions known to be critical for its biological function. The protein also includes a previously characterised J domain that although conformationally influenced by the residue extension, retains its folded state unlike the majority of the protein sequence.     

Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.

To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.     

Amplification mediated by polyomavirus large T antigen defective in replication.

The polyomavirus large T antigen promotes homologous recombination at high rates when expressed in rat cells carrying the viral replication origin and two repeats of viral DNA sequences stably integrated into the cellular genome. Recombination consists of both reciprocal and nonreciprocal events and is promoted by mutants defective in the initiation of viral DNA synthesis (L. St-Onge, L. Bouchard, and M. Bastin, J. Virol. 67:1788-1795, 1993). We have extended our studies to a rat cell line undergoing amplification of the viral insert. We show that large T antigen promotes amplification independently of its replicative function but that its origin-specific DNA binding activity is not sufficient to promote homologous recombination.     

Cyclin-dependent kinase regulation of the replication functions of polyomavirus large T antigen.

The amino-terminal portion of polyomavirus (Py) large T antigen (T Ag) contains two phosphorylation sites, at T187 and T278, which are potential substrates for cyclin-dependent kinases (CDKs). Our experiments were designed to test whether either or both of these sites are involved in the origin DNA (ori DNA) replication function of Py T Ag. Mutations were generated in Py T Ag whereby either or both threonines were replaced with alanine, generating T187A, T278A, and double-mutants (DM [T187A T278A]) mutant T Ags. We found that the Py ori DNA replication functions of T278A and DM, but not T187A, mutant T Ags were abolished both in vivo and in vitro. Consistent with this finding, it was shown that the ori DNA binding and unwinding activities of mutant T278A Py T Ag were greatly impaired. Moreover, whereas wild-type Py T Ag is an efficient substrate for phosphorylation by cyclin A-CDK2 and cyclin B-cdc2 complexes, it is phosphorylated poorly by a cyclin E-CDK2 complex. In contrast to mutant T187A, which behaved similarly to the wild-type protein, T278A was only weakly phosphorylated by cyclin B-cdc2. These data thus suggest that T278 is an important site on Py T Ag for phosphorylation by CDKs and that loss of this site leads to its various defects in mediating ori DNA replication. S- and G2-phase-specific CDKs, but not a G1-specific CDK, can phosphorylate wild-type T Ag, which suggests yet another reason why DNA tumor viruses require actively cycling host cells.     

Characterization of an immortalizing N-terminal domain of polyomavirus large T antigen.

Polyomavirus large T antigen has an N-terminal domain of approximately 260 amino acids which can immortalize primary cells but lacks sequences known to be required for DNA binding and replication. Treatment of full-length large T with either V8 protease or chymotrypsin yields an N-terminal fragment of 36 to 40 kDa and a C-terminal fragment of approximately 60 kDa. This finding suggests a division of the protein into two domains. Proteolysis experiments show that the N-terminal domain does not have strong physical association with the rest of the protein. It also does not self-associate. A construct expressing only the N-terminal 259 amino acids is sufficient for immortalization. The independently expressed N-terminal domain is multiply phosphorylated, although at a lower level than the same region in full-length large T. The 259-residue protein binds to both pRb and p107 with somewhat lower efficiency than the full-length protein.     

Expression of polyomavirus large T antigen by using a baculovirus vector.

A gene encoding the large T antigen of polyomavirus was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus so that gene expression was under the control of the strong, very late polyhedrin gene promoter. Significantly more large T antigen was produced in recombinant virus-infected insect cells than was observed in polyomavirus-transformed mouse cells. The insect-derived T antigen exhibited polyomavirus origin-specific DNA binding. The baculovirus expression system provides a convenient source of T antigen for in vitro studies.     

High-frequency recombination mediated by polyomavirus large T antigen defective in replication.

We investigated the mechanism by which the large T antigen (T-Ag) of both polyomavirus and simian virus 40 (SV40) promotes homologous recombination in mammalian cells. To this end, we constructed a rat cell line, designated Hy5, that carries two mutated copies of the polyomavirus middle-T-Ag (pmt) oncogene lying as direct repeats on the same chromosome. The structure of the viral insert was devised so that intrachromosomal recombination between the pmt repeats reconstitutes wild-type pmt and yields cell populations amenable to selection for the transformed phenotype. Correction of pmt by gene conversion occurred spontaneously at a rate of ca. 1.7 x 10(-7) per cell generation and was masked by another recombination event that also led to the transformation of the Hy5 cell line. This event was identified as chromosomal inversion and overexpression of the upstream pmt copy as a result of homologous recombination between adjacent pBR322 sequences. Both events were promoted by the polyomavirus large T-Ag by several orders of magnitude, as well as by mutants defective in the initiation of viral DNA synthesis. Large T-Ag also promoted reconstitution of wild-type pmt by unequal exchange between sister chromatids, yielding structures compatible with some of the chromosomal aberrations commonly observed in transformed cells. Our data indicate that large T-Ag has a recombination-promoting activity that can be dissociated from its replicative function.     

Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin.

We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

Zinc-binding and protein-protein interactions mediated by the polyomavirus large T antigen zinc finger.

Polyomavirus large tumor antigen (LT) contains a potential C2H2 zinc binding element between residues 452 and 472. LT also contains a third histidine in this region, conserved among the polyomavirus LTs. Synthetic peptides of this region bound a single atom of zinc, as determined by spectroscopic analysis. Blotting experiments also showed that fusion proteins containing the element, as well as full-length LT, bound 65Zn. Polyomavirus middle T and small T antigens also bound zinc in the blotting assay. Site-directed mutagenesis showed the importance of this element in LT. Point mutations in four of the conserved residues (C-452, C-455, H-465, and H-469) blocked the ability of LT to function in viral DNA replication, while mutation of H-472-->L decreased replication to 1/30th that of the wild type. Point mutations in intervening residues tested had little effect on replication. Mutants resulting from mutations in the conserved cysteine or histidine residues retained the ability to bind origin DNA. However, they did show a defect in self-association. Because double-hexamer formation is involved in DNA replication, this deficiency is sufficient to explain the defect in replication. Mutants created by point mutations of the coordinating residues were also deficient in replication-associated phosphorylations.     

Gene targeting in rat embryo fibroblasts promoted by the polyomavirus large T antigen.

We used the recombination-promoting activity of the polyomavirus large T antigen (T-ag) to increase the frequency of gene targeting in rat fibroblasts. We constructed a cell line carrying a functional polyomavirus replication origin and a transformation-defective middle T-ag oncogene. The structure of the locus was such that homologous recombination with the targeting DNA reconstituted a functional transforming gene and converted the cells from the normal to the transformed state. Introduction of the large T-ag with the targeting DNA promoted recombinational events that corrected the mutation in either the target locus or the targeting DNA. The frequency of recombination was not substantially influenced by the extent of homology between the recombining sequences. However, it was reduced when the replication origin was inactivated in the targeting DNA, and was reduced further when the origin was inactivated in the target locus.     

J domain-independent regulation of the Rb family by polyomavirus large T antigen

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb(-) and J(-) LTs revealed additional differences. Wild-type but not Rb(-) LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.     

Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen.

Essential nucleotide contacts between the polyomavirus large T antigen and its multiple specific binding regions within the regulatory sequences of the polyomavirus genome were determined in vitro by methylation interference. Methylation of any of the guanine residues of the 5'-G(A/G)GGC-3' pentanucleotide repeats in large-T-antigen-binding regions A, B, C, and 3 (A. Cowie and R. Kamen, J. Virol. 52:750-760, 1984) interfered with T antigen binding. Within regions A, B, and C these pentanucleotides are spaced 5 or 6 base pairs apart. Therefore, the clusters of contacted nucleotides within each of these binding regions are localized along one face of the DNA helix. Methylation of guanines within the sequences between the pentanucleotide repeats did not interfere with binding. The ORI binding region contains four additional pentanucleotide sequences within a region of dyad symmetry. Methylation of only particular guanines of these pentanucleotides interfered with T antigen binding. The spatial arrangement of the pentanucleotides in the ORI is such that the clusters of contacted guanines are situated around the DNA helix, thereby forming a very different arrangement from that found in the other binding regions. A model is discussed in which cooperative interactions between T antigen protomers, recognizing individual pentanucleotides, determines the strength and the function of different T antigen-DNA interactions.     

Natural biology of polyomavirus middle T antigen.

"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.     

Elements of the polyomavirus replication origin required for homologous recombination mediated by large T antigen.

We introduced various elements of the polyomavirus origin of DNA replication into the genome of rat cells, and we analyzed their capacity to elicit rearrangements within the integrated sequences when exposed to large T antigen. The cis-acting sequences required for homologous recombination were those that make up a functional replication origin.