The conserved core enzymatic activities and the distinct dynamics of polyomavirus large T antigens.

Several human polyomaviruses including JCV, BKV and TSV are associated with diseases, particularly in immunosuppressed patients. While the large T antigen (LT) encoded by the monkey polyomavirus SV40 is well studied, and possesses intrinsic ATPase and DNA helicase activities, the LTs of the human polyomaviruses are relatively uncharacterized. In order to evaluate whether these enzymatic activities, which are required for viral DNA replication, are conserved between polyomaviruses, we performed a comparative study using the LTs from JCV, TSV and SV40. The ATPase and DNA helicase activities and the interaction with the cellular tumor suppressor p53 were assayed for the purified Zn-ATPase domains of the three LTs. We found that all Zn-ATPases were active ATPases. The Zn-ATPase domains also functioned as DNA helicases, although the measured kinetic constants differed among the three proteins. In addition, when tested against four small molecule ATPase inhibitors, the Zn-ATPase domains of TSV was more resistant than that of SV40 and JCV. Our results show that, while LTs from JCV and TSV share the core ATPase and DNA helicase activities, they possess important functional differences that might translate into their respective abilities to infect and replicate in hosts.

High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity.

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC(50), cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC(50) of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.

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.

RNA polymerase II pauses in vitro, but does not terminate, at discrete sites in promoter-proximal regions on polyomavirus transcription complexes.

Many RNA polymerases stall and/or prematurely terminate transcription nearby the early and late promoters of polyomavirus in vivo during the late phase of productive infection. In this paper we analyzed the RNAs made when these promoter-proximal RNA polymerases were allowed to elongate their nascent chains in vitro on viral transcription complexes isolated from infected cells. RNA was labeled in the presence of a high specific activity of one [alpha-32P]-ribonucleoside triphosphate (rNTP) (less than 1 microM final concentration) and saturating concentrations of the other three rNTPs. Under these conditions, promoter-proximal RNAs of discrete sizes were produced. We show that these discrete RNA species are produced by pausing of RNA polymerase II due to limiting concentrations of one of the rNTPs, and that the positions of the pause sites depend on which rNTP is limiting. This pausing does not result in release of the RNA polymerase or the nascent RNA chain from the transcription complex, as these chains can be further extended when high concentrations of all four rNTPs are supplied. Our results conflict with the interpretations of other investigators who suggest that formation of discrete RNA products, under similar in vitro conditions, reflects authentic termination by RNA polymerase II.

Recombinant retroviruses that transduce middle T antigen cDNAs derived from polyomavirus mutants: separation of focus formation and soft-agar growth in transformation assays and correlations with kinase activities in vitro.

To study correlations between cellular transformation and the biochemical properties of polyomavirus middle T antigen, middle T cDNAs have been derived from the polyomavirus mutants dl1015, dl23, and NG59b and have been introduced into rodent fibroblast cell lines by using a retrovirus vector. It was found that all three mutants are completely defective in inducing growth in soft agar but possess a range of activities in assays of focus formation on cell monolayers. Furthermore, when assays of middle T antigen-associated kinase activities were performed in vitro, a correlation between the level of associated phosphatidylinositol kinase activity and the ability of mutant middle T antigens to induce focus formation was observed. However, the association of this activity with middle T antigen does not appear to be sufficient to bring about full transformation, since the middle T antigen derived from dl1015 is completely defective for soft-agar growth but is associated with a level of phosphatidylinositol kinase activity which is comparable to that of the wild type. Therefore, some other unidentified middle T antigen function may also be required for full transformation.

Analysis of middle tumor antigen and pp60c-src interactions in polyomavirus-transformed rat cells.

The relative abundance of pp60c-src molecules associated with polyomavirus (Py) middle tumor antigen (MTAg) and the relative abundance of MTAg associated with pp60c-src in a variety of Py-transformed rat cells was determined by quantitative immunoblot analyses which detect pp60c-src or Py MTAg. The results demonstrate that approximately 5 to 10% of the total immunoprecipitable pp60c-src molecules in Py-transformed rat cells are stably associated with MTAg and have elevated protein kinase activities. In these same cells, it was found that approximately 10 to 15% of the detectable MTAg molecules are stably associated with pp60c-src. Other results presented in this report demonstrate that approximately 50 to 75% of the total MTAg-associated cellular tyrosine kinase activity potentially represents the enzymatic activity of pp60c-src, while the remaining 25 to 50% represents the activity of other cellular tyrosine kinases. Our results also show that most pp60c-src molecules associated with Py MTAg do not possess electrophoretic mobilities that are altered from those of pp60c-src molecules not associated with MTAg or pp60c-src molecules obtained from normal rodent cells.

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity.

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.

Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens.

The location and sequence composition of a consensus element of the nucleotide binding site in both simian virus 40 (SV40) and polyomavirus (PyV) large tumor antigens (T antigens) can be predicted with the assistance of a computer-based pattern-matching system, ARIADNE. The latter was used to optimally align elements of T antigen primary sequence and predicted secondary structure with a "descriptor" for a mononucleotide binding fold. Additional consensus elements of the nucleotide binding site in these two proteins were derived from comparisons of T antigen primary and predicted secondary structures with x-ray structures of the nucleotide binding sites in four otherwise unrelated proteins. Each of these elements was predicted to be encompassed within a 110-residue segment that is highly conserved between the two T antigens residues 418-528 in SV40 T antigen and residues 565-675 in PyV). Results of biochemical and immunologic experiments on the nucleotide binding behavior of these proteins were found to be consistent with these predictions. Taken together, the latter have resulted in a topological model of the ATP binding site in these two oncogene products.

Characterization of middle T antigen expressed by using an adenovirus expression system.

The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60c-src. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60c-src remained unassociated. Immunoaffinity purification resulted in approximately 100 micrograms of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74- and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.

Analysis of polyomavirus middle-T-antigen-transformed rat cell variants expressing different levels of pp60c-src.

We characterize two independent variant cellular clones which arose following in vitro passage of polyomavirus middle-T-antigen (MTAg)-transformed FR3T3 cells expressing RNA complementary to c-src mRNA. These clones were initially flat and underwent morphologic transformation at a high frequency to a phenotype indistinguishable from that of parental MTAg-transformed FR3T3 cells. Biochemical analysis of the flat clones prior to phenotypic conversion revealed that these cells synthesized little detectable pp60c-src and had correspondingly low levels of pp60c-src protein kinase activity and MTAg-associated protein kinase activity. The flat cell clones did not possess detectable focus-forming activity, were not capable of detectable anchorage-independent growth, and had saturation densities and doubling times below those normally observed for FR3T3 cells. Following conversion of the flat clones to a shape resembling that of typical MTAg-transformed cells, the abundance of pp60c-src, pp60c-src kinase activity, and MTAg-associated in vitro protein kinase activity were all restored to the levels found in the parental MTAg transformants. These cells had growth rates, focus-forming activities, anchorage-independent growth rates, and saturation densities similar to those of the parental MTAg-transformed rat cells. These data provide additional evidence that maintenance of a transformed phenotype by polyomavirus MTAg in established rat cell lines depends, at least in part, on a minimal threshold level of pp60c-src.

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.

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.

Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant.

A modular gene with a cDNA encoding the polyomavirus middle T antigen positioned behind the adenovirus type 2 major late promoter and tripartite leader was substituted for the E1a region in an adenovirus vector. Permissive human cells infected with this recombinant produce middle T protein at levels as high as those of the most abundant late adenoviral proteins, e.g., hexon or fiber. This level represents at least a 40-fold increase over that observed in a polyomavirus lytic infection of murine cells. Partial proteolytic mapping showed that this protein has the same primary structure as middle T protein produced in polyomavirus-infected murine cells. The adenovirus recombinant-generated middle T protein exhibited in vitro kinase activity, although at an approximately 10-fold-lower specific activity than that of middle T protein from polyomavirus-infected murine cells. Comparison of the expression levels of this middle T antigen-containing adenovirus vector with a similar construction encoding dihydrofolate reductase suggested that the translation efficiency of the inserted gene was dependent upon the proximity of its initiation codon to the tripartite leader. We tested this possibility by comparing three dihydrofolate reductase recombinants among which the spacing between the initiation codon and tripartite leader varied from 188 to 36 nucleotides. The efficiency of expression of dihydrofolate reductase protein dramatically increased as this spacing was reduced.

Overproduction of polyomavirus middle T antigen in mammalian cells through the use of an adenovirus vector.

To overproduce biologically active polyomavirus middle T antigen, we used an adenovirus vector and human 293 cells as hosts. Two helper-independent recombinant adenoviruses were isolated that contain a hybrid transcription unit, in differing orientations, at a site in the adenovirus genome from which the E1a and most of the E1b transcription units have been deleted. The hybrid transcription unit consists of the adenovirus type 2 major late promoter and tripartite leader and a cDNA segment capable of encoding polyomavirus middle T antigen and accompanying 3' RNA-processing signals. Both recombinant viruses were stable and replicated to high titers in human 293 cells. The polyomavirus sequences were expressed, predominantly at late times after infection of 293 cells, to yield mRNAs that encoded middle T antigen. One of the recombinant viruses also expressed a middle T antigen-related protein in 293 cells. The latter was translated from one of several novel mRNA species that resulted from aberrant splicing and incomplete RNA processing of precursor RNA transcripts. Comparison of the amount of middle T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with either of the recombinant adenoviruses, under optimal conditions for each system, revealed at least a 10-fold greater yield of the protein on a per-cell basis in the latter system than in the former. The recombinant-virus-encoded middle T antigen was biologically active, as evidenced by its ability to associate with and serve as a substrate for human pp60c-src. The functionality of the middle T antigen was further confirmed by demonstrating that both recombinant viruses efficiently transformed Rat-1 cells. These recombinant viruses will be useful to overproduce middle T antigen and to introduce the polyomavirus oncogene into a wide variety of mammalian cells.

Biochemical properties associated with the immortalizing domain of the large T protein of polyoma virus.

A fragment of polyoma virus DNA lacking the carboxy-terminal part of the large T antigen coding region is sufficient to express the functions of the entire gene in cell transformation. Two cell lines expressing this truncated DNA were studied, one of them producing a large T-related protein of the expected size (37 kDa) and the other one, a shorter product (34 kDa). Both proteins were phosphorylated and localized in the nucleus, but devoid of ATPase and nucleotide binding activities. As the complete protein, the larger product, but not the shorter variant, exhibited sequence-specific DNA binding properties. These results indicate that ATPase and nucleotide-binding activities are not required for immortalization, and suggest that recognition of specific DNA sequences may be dispensable.

Regulation of viral and cellular promoter activity by polyomavirus early proteins.

The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen.

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.

The expression and properties of polyoma virus middle-T antigen in simian cells.

SV40 late replacement vectors containing the polyoma middle-T coding sequences have been constructed. Mixed hybrid virus stocks have been obtained through complementation with a defective SV40 helper genome (dl 1055) following DNA transfection into CV-1 cells. Middle-T antigen is expressed in the infected simian cells at about 5-10 fold higher levels than in polyoma virus-infected mouse cells and has the pp60c-src-associated tyrosine-specific protein kinase activity in vitro. However, the 'specific activity' of the kinase in extracts of the infected CV-1 cells is lower than that observed in polyoma infected 3T6 cell extracts. The half-life of middle-T antigen in the CV-1 cells is about 4 h but the in vitro kinase activity associated with middle-T has a half-life of at least 8 h and hence appears to be stabilized. The in vivo phosphorylated species of middle-T has been shown by sucrose gradient analyses to be largely distinct from the middle-T with associated protein kinase activity in vitro.

A frameshift at a mutational hotspot in the polyoma virus early region generates two new proteins that define T-antigen functional domains.

A frameshift mutation, arising from the deletion of any one of nine consecutive cytidines in the region of Py DNA encoding both the midregion of large T-Ag and the C-terminal region of middle T-Ag, yields unstable flat cell revertants that synthesize two novel viral proteins in which shuffling of the different domains of the Py T-Ags has occurred. The first protein (37 kd) is a hybrid containing the N-terminus of large T-Ag and the hydrophobic C-terminus of middle T-Ag. The latter domain is responsible for membrane association, even in the 37 kd hybrid protein. The second protein (43 kd), which contains the N-terminal 75% of middle T-Ag, has an associated protein kinase activity and forms a complex with c-src, but cannot induce a transformed phenotype.