DNA polymerase activities in growing cells infected with simian virus 40.

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Cellular proteins which can specifically associate with simian virus 40 small t antigen.

When crude, radiolabeled extracts of various cells were applied to homogeneous simian virus 40 small t antigen-Sepharose adsorbents, three cell proteins (57, 32, and 20 kilodaltons [kDa]) bound specifically. Each also bound to an insoluble, truncated t derivative composed of the COOH-terminal 123 residues of the protein. The binding of these proteins was greatly inhibited after reduction and alkylation of the t ligand. Therefore, some element of native conformation, but not all of the primary structure of t, is necessary for this binding property, which may constitute a discrete, in vitro biochemical function of this protein. Results of cell fractionation experiments suggested that the 57- and 32-kDa proteins are nonnuclear cell constituents, whereas the 20-kDa protein was closely associated with a detergent-washed nuclear fraction. Specific immunoblotting and comparative partial proteolytic digestion analyses indicated that the 57-kDa protein is tubulin, a major component of the cytoskeleton. In this regard, t and tubulin were observed to coimmunoprecipitate from crude cell extracts after incubation with monospecific anti-t antibody. Therefore, it is possible that t and tubulin interact in vivo.     

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Collagenase production by immortalized human aortic endothelial cells infected with simian virus 40.

Human aortic endothelial cells, isolated at autopsy from a 52-year-old male dying from lung cancer, were treated with simian virus 40 (SV40). One colony was isolated from the infected endothelial cell culture 4 weeks after infection. The cells expressed SV40 large T antigen and p53 protein (p53) in their nuclei but lacted the characteristics of a transformed phenotype. The cells grew well in a monolayer over the 97th passage and exhibited Factor VIII-related antigen, Ulex europaeus 1 agglutinin (UEA-1) as endothelial cell markers, and a well-developed fibronectin network. The amount of prostacyclin synthesized by the cells was less than the amount synthesized by normal aortic or umbilical cord vein endothelial cells. The cells produced relatively large amounts of procollagenase, and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) augmented the ability of the cells to produce this enzyme. These immortalized human aortic endothelial cells, which have some characteristics of normal endothelial cells and, like capillary endothelial cells, have the ability to produce collagenase, will probably prove useful for studies of atherosclerosis and angiogenesis.     

Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin.

Detergent extraction of simian virus 40 (SV40) DNA from infected monkey CV-1 cells, after a brief exposure to the drug camptothecin, yields covalent complexes between topoisomerase I and DNA that band with reduced buoyant densities in CsCl. The following lines of evidence indicate that the enzyme is preferentially associated with SV40 replicative intermediates. First, the percentage of the isolated labeled viral DNA that exhibited a reduced buoyant density is inversely proportional to the length of the labeling period and approximately parallels the percentage of replicative intermediates for each labeling time (5 to 60 min). Second, after labeling for 60 min, the isolated low-density material was found to be enriched for replicative intermediates as measured by sedimentation in neutral sucrose. Third, analysis of extracted viral DNA by equilibrium centrifugation in CsCl-propidium diiodide gradients that separate replicating molecules from completed form I DNA revealed that camptothecin pretreatment specifically caused the linkage of topoisomerase I to replicating molecules. In addition, analysis of the low-density material obtained under conditions when only the newly synthesized strands of the replicative intermediates were labeled showed that the enzyme was associated almost exclusively with the parental strands. Taken together, these observations indicate that topoisomerase I is involved in DNA replication, and they are consistent with the hypothesis that the enzyme provides swivels to allow the helix to unwind. The observed bias in the distribution of topoisomerase I on intracellular SV40 DNA could be the result of rapid encapsidation of replicated molecules that precludes the association of topoisomerase I with the DNA or, alternatively, the result of a specific association of the enzyme with replicative intermediates.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.     

Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells.

Two cellular proteins are found to be complexed with simian virus 40 small-t antigen in cellular extracts. The complex is a relatively unstable but dynamic one which can dissociate and reform in extracts. In extracts of permissive monkey kidney cells, the small-t antigen appeared to be present in excess, whereas the cellular proteins were nearly entirely committed to the complex in permissive monkey kidney cells.     

Effect of hypertonic conditions on protein synthesis in cells productively infected with simian virus 40.

Hypertonic medium selectively suppressed the synthesis of most host cell polypeptides relative to the synthesis of simian virus 40 capsid polypeptides and a minority of cellular polypeptides, notably histones. Under optimal hypertonic conditions, the synthesis of the major capsid polypeptide (VP1) is enhanced about sevenfold relative to host polypeptide synthesis. Because of the small amounts of the other nonhistone capsid polypeptides (VP2) and VP3) present in cell lysates, it was difficult to quantitate the extent, if any, of their enhancement. The maintenance of the restricted pattern of protein synthesis caused by hypertonic medium was dependent on continual peptide chain initiations. The resistance of viral protein synthesis to hypertonic conditions provides a means of detecting relatively low levels of intracellular viral protein synthesis. Analysis of the specific activity of the acid-soluble [3H]lysine pool indicated that the rate of incorporation of [3H]lysine into protein was an overestimation of the actual rate of overall protein synthesis occurring in cells exposed to hypertonic as compared to isotonic conditions. Since it is likely that both cellular and viral protein synthesis draw lysine from a single pool, this change in pool specific activity does not affect the analysis of relative rates of protein synthesis at a given level of tonicity.     

Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40.

T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.     

Collagenase production by immortalized human aortic endothelial cells infected with simian virus 40

Human aortic endothelial cells, isolated at autopsy from a 52-year-old male dying from lung cancer, were treated with simian virus 40 (SV40). One colony was isolated from the infected endothelial cell culture 4 weeks after infection. The cells expressed SV40 large T antigen and p53 protein (p53) in their nuclei but lacted the characteristics of a transformed phenotype. The cells grew well in a monolayer over the 97th passage and exhibited Factor Vlll-related antigen, Ulex europaeus 1 agglutinin (UEA-1) as endothelial cell markers, and a well-developed fibronectin network. The amount of prostacyclin synthesized by the cells was less than the amount synthesized by normal aortic or umbilical cord vein endothelial cells. The cells produced relatively large amounts of procollagenase, and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) augmented the ability of the cells to produce this enzyme. These immortalized human aortic endothelial cells, which have some characteristics of normal endothelial cells and, like capillary endothelial cells, have the ability to produce collagenase, will probably prove useful for studies of atherosclerosis and angiogenesis.     

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.     

Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses.

The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2-infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ND2, Ad2+ND4, and Ad2+ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.     

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.     

A malignant astrocytoma containing simian virus 40 DNA in a macaque infected with simian immunodeficiency virus

Abstract: Polyomaviruses have proven oncogenicity in nonhost experimental animals; however, studies concerning the association between human brain tumors and simian and human polyomaviruses have yielded inconclusive results. We examined the relationship of SV40 to a malignant astrocytoma found in the right frontal lobe of a pigtail macaque (Macaca nemestrina) infected with simian immunodeficiency virus (SIV). Consistent with the histologic diagnosis, the tumor was immunoreactive with antibodies to S-100 protein, vimentin, and glial fibrillary acidic protein, but negative for neurofilament protein, synaptophysin, neuron-specific enolase, and chromogranin A. At the time of SIV inoculation, the animal was seropositive for SV40. Polymerase chain reaction assay of tumor DNA, but not normal brain DNA, yielded a 300 base-pair fragment corresponding to the carboxy-terminal coding region (C-terminus) of the large T antigen gene of SV40, suggesting an association with the tumor.     

Cellular and cell-free synthesis of simian virus 40 T-antigens in permissive and transformed cells.

mRNA extracted from a variety of simian virus 40 (SV40)-infected monkey cell lines directs the cell-free synthesis of viral T-antigen polypeptides with molecular weights estimated as 90,000 and 17,000. However, the size, abundance, and distribution of these T-antigens synthesized in vivo vary greatly over a range of permissive and transformed cell lines. To establish whether differences in the size of T-antigen polypeptides can be correlated with the transformed or lytic state, recently developed lines of SV40-transformed monkey cells that are permissive to lytic superinfection were analyzed for T-antigen. In these cells, regardless of the state of viral infection, the size and pattern of T-antigen are the same. However, species differences in the largest size of T-antigen are the same. However, species differences in the largest size of T-antigen do exist. In addition to the 90,000 T-antigen, mouse SV3T3 cells contain a 94,000 T-antigen polypeptide as well. Unlike the size variations in monkey cells, which are due to modification of T-antigen polypeptides, the 94,000 SV3T3 T-antigen results from an altered mRNA, since the cell-free products of SV3T3 mRNA also contains the 94,000 T-antigen polypeptide.     

Presence in growth-arrested cells of cellular proteins that interact with simian virus 40 small-t antigen.

Two cellular proteins, 56K and 32K, found in association with simian virus 40 small-t antigen were not induced by viral infection. In addition, the proteins were expressed by cells in the growth arrest period, a time in which small-t function is of importance in infection and transformation.