ATP phosphohydrolase (ATPase) activity of a polyoma virus T antigen

Among the various polyoma virus T antigens which have so far been identified, only the large-T and a 63 000-Mr polypeptide were found to bind to double-stranded calf thymus DNA. The proteins were not retained on single-stranded DNA-cellulose columns, and a purification procedure was designed on the basis of this observation. Purified fractions (approx. 1000-fold) exhibited an enzymatic activity which converts ATP into ADP and Pi. This activity was quantitatively inhibited after preincubation in the presence of anti-(polyoma T antigen) immunoglobulins and was shown to be dependent on a virus-coded gene product (alpha gene) on the basis of the following observations: (a) ATPase activity from cells infected with tsa mutants of polyoma was reduced after a shift to the restrictive temperature; (b) the enzyme purified from tsa-infected cells maintained at the permissive temperature was more thermolabile in vitro than that prepared in parallel from cells infected with wild-type virus.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication II. Behavior During Incubation of Isolated Nuclei

The synthesis of polyoma DNA was studied in isolated nuclei from hydroxyurea-inhibited 3T6 cells infected with polyoma virus. During incubation of nuclei under conditions suitable for polyoma DNA synthesis in vitro, the short DNA fragments with a sedimentation coefficient of 4S formed in vivo (hydroxyurea fragments) became associated with preformed, replicating DNA strands. Centrifugation in dye-buoyant density gradients showed that the fragments formed part of the structure of the replicative intermediate of polyoma DNA. The proportion of "young" replicative intermediates was larger after hydroxyurea inhibition than in uninhibited controls. Hydroxyurea fragments appear to be closely related to the 4S fragments formed as normal intermediates during discontinuous synthesis of polyoma DNA.     

Salt-stable binding of the large T protein to DNA in polyoma virus chromatin.

Nucleoprotein complexes extracted from the nuclei of mouse cells lytically infected with polyoma virus contain an ATPase activity which appears to correspond to that of the viral large T protein, as it exhibits the same characteristic properties; in particular, the activity is extensively inhibited by polyclonal antibodies from animals bearing polyoma tumors (anti-T antigen antibodies) and by monoclonal antibodies against large T. Significant amounts of DNA were immunoprecipitated by adding these antibodies to the nucleoprotein complex, suggesting that the protein is tightly bound to DNA in the viral chromatin. Since one of the monoclonal antibodies quantitatively immunoprecipitated the pulse-labeled replicative intermediates, we conclude that some large T protein remains physically associated with the DNA throughout its replication cycle. After exposure to salt concentrations higher than 1 M KCl, about half of the large T-specific ATPase activity was still observed to co-sediment with 21S form I viral DNA. The observations that the sedimentation coefficient of the salt-stable complexes was shifted to 16S after a limited endonucleolytic digestion, and that both the viral DNA and the ATPase activity were co-precipitated in the presence of polyethylene glycol at high ionic strength, further demonstrated that the protein is engaged in an unusually stable complex with DNA in the viral chromatin.     

Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses

Incubation of crude extracts from cells lytically infected with polyoma virus in the presence of periodate-oxidized [alpha-32P]ATP led to the radioactive labeling of one main polypeptide immunoprecipitated by anti-T antigen antibodies. It was absent from extracts of mock-infected cells and exhibited an apparent Mr value of 105,000, identical with that of the large-T viral protein. This polypeptide was unambiguously identified as large-T on the basis of the heat sensitivity of the in vitro labeling in extracts from cells infected with a tsa mutant. The amount of incorporated radioactivity was found to increase linearly with that of infected cell extract and with the incubation time. Labeling resulted from the specific binding of an oxidized ATP molecule to a nucleotide binding site of the large-T protein, since it was efficiently completed by unlabeled ATP. Study of the dependence on the concentration of oxidized ATP evidenced a first order kinetic for the labeling reaction. Similar results were obtained for the large-T protein of SV40 virus.     

Further characterization of the phenotype of ts20, a DNAts mutant of BALB/3T3 cells

Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 degrees to 39 degrees C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 degrees C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 degrees C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I.     

Multiple Free Viral DNA Copies in Polyoma Virus-Transformed Mouse Cells Surviving Productive Infection

Mouse 3T6 cells were infected with polyoma virus at high multiplicity, and survivors were isolated. Clones from single cells were then established and were found to be resistant to a second infection. However, in some clones viral functions could at least be partially expressed during reinfection, as judged from a stimulation of nuclear tumor antigen expression. One such clone was studied in detail. These cells were transformed and produced low amounts of virus (less than 1 PFU per cell per generation). The persistent infection did not seem to be a carrier-state phenomenon, since infectious-center assays showed that most cells produced virus. The resistance of the cells to reinfection can be explained by interference from viral DNA present in the cells, averaging about 1,500 "free" copies per cell. This DNA had the normal physical characteristics of polyoma DNA. However, it had a slightly larger size than authentic polyoma DNA. Mapping with restriction endonucleases showed that the addition to the DNA was about 5% of the wild-type genome and was located close to the origin of DNA replication. This DNA was infectious, although it had a 10-fold lower infectivity than wild-type polyoma DNA. Both virus and DNA from the polyoma-resistant cells had a small-plaque morphology, as opposed to the large-plaque morphology of the virus used for the initial selection of cells.     

Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.     

Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.     

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.     

Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus.

A fibroblast line of the 3T3 type with a low saturation density was established from Fisher rat embryo cells. After infection with either wild-type or tsa mutant polyoma virus, transformants were isolated and cloned at 33 degrees C on the basis of their ability either to grow as dense foci on plastic in liquid medium (type N) or to form colonies in soft agar (type A). Polyoma T antigen was detected in all of the transformed lines. The following growth characteristics were studied for both types at 33 and 41 degrees C: saturation density, growth in soft agar and at a low serum concentration, colony-forming ability, and generation time. tsa-N transformants behaved at 33 degrees C similarly to transformed cells, but reverted at 41 degrees C to the nontransformed phenotype for all of these characters. tsa-A transformants and all of the wild-type transformants exhibited the transformed phenotype at both low and high temperatures. These results led us to distinguish at least two types of virus-induced transformants. In one of them, the activity of the protein affected by the tsa mutation appears to be necessary for the expression of several of the characters defining the transformed state.     

Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection.

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.     

Deoxyuridine triphosphate pools after polyoma virus infection

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.     

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.     

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.     

Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: removal of early region introns from polyoma virus mutants

We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37 degrees C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.     

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.