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.     

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.     

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.     

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.     

Intrachromosomal recombination mediated by papovavirus large T antigens.

To investigate the mechanism by which the large T antigen (T-Ag) of polyomavirus and simian virus 40 can promote recombination in mammalian cells, we analyzed homologous recombination events occurring between two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functional pmt gene by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. Introduction of the polyomavirus large T (plt) oncogene into the cell line by DNA transfection promoted recombination very efficiently, with rates in the range of 10(-1) to 10(-2) per cell generation. Recombination was independent of any amplification of viral sequences and could even be promoted by the large T-Ag from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large T-Ag, we propose a novel mechanism of nonconservative recombination involving slipped-strand mispairing between the two viral repeats followed by gap repair synthesis.     

The replication functions of polyomavirus large tumor antigen are regulated by phosphorylation.

Polyomavirus (Py) large T antigen (T Ag) contains two clusters of phosphorylation sites within the amino-terminal half of the protein. To characterize possible regulatory effects of phosphorylation on viral DNA replication, Py T Ag was treated with calf intestinal alkaline phosphatase (CIAP). Incubation of the protein with a range of phosphatase concentrations caused progressive loss of phosphate without affecting its stability. Treatment with smaller quantities of CIAP stimulated the ability of the viral protein to mediate replication of constructs containing the viral replication origin, while higher concentrations of CIAP caused a marked diminution of this replication function. Several biochemical activities of Py T Ag were examined after CIAP treatment. Py T Ag DNA unwinding and nonspecific DNA binding were only slightly affected by dephosphorylation. However, as determined by DNase I footprinting experiments, treatment with smaller amounts of CIAP stimulated specific binding to the Py replication origin by Py T Ag, while treatment with larger amounts of CIAP caused marked inhibition of origin-specific binding by the viral protein. Phosphotryptic maps of Py T Ag before or after treatment with CIAP revealed changes in individual phosphopeptides that were uniquely associated with either the stimulation or the inhibition of replication. Our data therefore suggest that Py T Ag is regulated by both repressing and activating phosphates.     

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.     

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.     

Localization of the phosphorylations of polyomavirus large T antigen.

Polyomavirus large T antigen is phosphorylated on both serine and threonine residues at a ratio of approximately 6 to 1. This phosphorylation could be resolved into a series of nine Staphylococcus aureus V8 phosphopeptides. All of these were found in an N-terminal chymotryptic fragment with a molecular weight of 57,000. A C-terminal formic acid fragment of 50,000-molecular-weight lacked phosphate. Therefore, unlike simian virus 40 large T antigen, polyomavirus large T antigen has no significant C-terminal phosphorylation. Limited V8 and hydroxylamine cleavage showed that the phosphorylations can be localized to two different portions of the molecule. A significant fraction of the phosphate was localized in the N-terminal portion of the molecule before residue 183. Within this region V8 peptides 4, 8, and 9 represented phosphorylations that were more proximal, while peptides 1, 2, and 3 included more distal phosphorylations. None of these phosphorylations appeared analogous to those of simian virus 40 large T antigen. V8 phosphopeptides 5 and 7 were more distal and could be distinguished in biological experiments from the N-terminal phosphorylations. Formic acid mapping suggested that much, if not all, of this phosphorylation is located between residues 257 and 285.     

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.     

Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen.

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.     

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.     

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.     

The retinoblastoma protein alters the phosphorylation state of polyomavirus large T antigen in murine cell extracts and inhibits polyomavirus origin DNA replication

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.     

Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication.

Polyomavirus DNA replication is normally restricted to rodent cells, and simian virus 40 (SV40) DNA replication is restricted to primate cells. We demonstrate that DNAs containing the polyomavirus origin can be replicated in monkey cells which constitutively express SV40 large T antigen. Permissivity is most likely caused by SV40 T antigen modification of cellular protein(s) required to replicate the polyomavirus origin. A possible target for the T-antigen-induced modification is DNA polymerase alpha-DNA primase.     

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.     

High-frequency homologous recombination in plants mediated by zinc-finger nucleases

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.     

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.