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.     

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.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

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.     

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.     

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.     

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.     

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.     

Replication-dependent transactivation of the polyomavirus late promoter.

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.     

Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication.

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the cis-acting origin of DNA replication, with its auxiliary DNA elements, and the trans-acting viral large tumor antigen (T antigen). Appropriate interactions between them initiate the assembly of a replication complex which, together with cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of cis-acting elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however, these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to initiate replication at the origin of another virus. We have studied the basis for such replication selectivity between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro. However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA replication can evolve replication selectivity and by which the function of diverse cellular origins might be temporally activated during the S phase of the eukaryotic cell cycle.     

Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen.

Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed.