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.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts.

Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild-type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.     

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen.

To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1-521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyT1-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens.     

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.     

Simultaneous overexpression of avian pp60c-src and polyomavirus middle T antigen in mammalian cells.

Recombinant adenoviruses bearing the avian c-src gene and polyomavirus middle-T-antigen gene were isolated and used to simultaneously overexpress both proteins in human 293 cells. Cells overexpressing both proteins had greater middle-T-antigen-associated tyrosine kinase activity than cells overexpressing only middle T antigen. By contrast, the intrinsic pp60c-src tyrosine kinase activity was not greater in cells overexpressing both proteins than in cells overexpressing only pp60c-src. This system of simultaneous overexpression provides a means of obtaining large quantities of pp60c-src, middle T antigen, and the complex between them.     

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.     

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.     

A mathematical model of homologous recombination in cultured cells.

This work presents a model describing the rate of recombination between homologous segments of DNA stably integrated into the genome of cultured cells. The model has been applied to rat cell lines carrying the polyomavirus middle T oncogene and a functional origin of viral DNA replication. Introduction of the gene coding for the polyoma large T antigen or the SV40 large T antigen into cells by DNA transfection promotes homologous recombination in the resident viral inserts with rates varying between 0.1 x 10(-3) and 3.7 x 10(-1) per cell generation.     

Stimulation of DNA replication from the polyomavirus origin by PCAF and GCN5 acetyltransferases: acetylation of large T antigen

The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.     

n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses.

Small DNA viruses are dependent on the interaction of early proteins (such as large T antigen) with host p53 and Rb to bring about the G1-to-S cell cycle transition. The large DNA viruses are less dependent on host regulatory genes since additional early viral proteins (such as viral DNA polymerase, DNA metabolic enzymes, and other replication proteins) are involved in DNA synthesis. A highly conserved domain of large T antigen (similar to the p53-binding region) exclusively identifies papovavirus, parvovirus, and papillomaviruses from all other larger DNA viruses and implies a conserved interaction with host regulatory genes. In this report, we show that 3 to 6 mM butyrate, a general cell cycle blocker implicated in inhibition of the G1-to-S transition, inhibits DNA replication of polyomavirus and human papillomavirus type 11 but not the replication of larger DNA viruses such as adenovirus types 2 and 5, herpes simplex virus type 1, Epstein-Barr virus, and cytomegalovirus, which all bypass the butyrate-mediated cell cycle block. This butyrate effect on polyomavirus replication is not cell type specific, nor does it depend on the p53 or Rb gene, as inhibition was seen in fibroblasts with intact or homozygous deleted p53 or Rb, 3T6 cells, keratinocytes, C2C12 myoblasts, and 3T3-L1 adipocytes. In addition, butyrate did not inhibit expression of polyomavirus T antigen. The antiviral effect of butyrate involves a form of imprinted state, since pretreatment of cells with 3 mM butyrate inhibits human papillomavirus type 11 DNA replication for at least 96 h after its removal. Butyrate, therefore, serves as a molecular tool in dissecting the life cycle of smaller DNA viruses from that of the larger DNA viruses in relation to the cell cycle.     

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.     

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.     

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.