-
Journal of Virology May 1983We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding...
We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.
Topics: Animals; Antigens, Viral; Antigens, Viral, Tumor; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; Mutation; Rats; Simian virus 40
PubMed: 6302314
DOI: 10.1128/JVI.46.2.475-480.1983 -
Journal of Virology May 1971To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide... (Comparative Study)
Comparative Study
To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. "Empty" virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.
Topics: Alkalies; Animals; Carbon Isotopes; Centrifugation, Density Gradient; Cesium; Chemical Precipitation; Chlorides; Chromatography, DEAE-Cellulose; Electrophoresis, Disc; Glycols; Haplorhini; Kidney; Mercaptoethanol; Molecular Weight; Peptides; Simian virus 40; Sodium; Sucrose; Sulfates; Tritium; Urea; Viral Proteins
PubMed: 4326753
DOI: 10.1128/JVI.7.5.635-641.1971 -
Journal of Virology Jun 1991Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in...
Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in culture. We have isolated immortalized derivatives (AR5 and HAL) from transformants generated with an origin-defective SV40 genome encoding a heat-labile large T protein (T antigen) and reported that both preimmortal and immortal transformants are continuously dependent on T antigen function for growth as determined by temperature shift experiments. In this study, we demonstrate complex formation between T antigen and the retinoblastoma susceptibility gene product (Rb) at 35 degrees C and observed a reduction in complexes under conditions of loss of T antigen function and growth inhibition at 39 degrees C. Viral oncogenes (polyomavirus large T protein and adenovirus E1A 12S protein) known to bind Rb were introduced into AR5 and HAL cells, both stably by gene transfer and transiently by virus vectors. Such double transformants are still unable to proliferate at 39 degrees C, although complex formation with the newly introduced oncogenes was demonstrated. We suggest that T antigen interacts with other cellular processes in addition to Rb to transform and immortalize human cells in culture. Our finding that p53-T antigen complexes are also temperature dependent in AR5 and HAL cells could provide such an additional mechanism.
Topics: Antigens, Viral, Tumor; Cell Cycle; Cell Line; Cell Transformation, Viral; Fibroblasts; Humans; Oncogenes; Retinoblastoma Protein; Simian virus 40; Temperature; Transfection
PubMed: 1851857
DOI: 10.1128/JVI.65.6.2845-2852.1991 -
Journal of Virology May 1986Simian virus 40 (SV40) large T antigen (TAg), both free and bound to mature 70S and replicating 90S SV40 chromosomes, was prepared from lytically infected cells. The...
Simian virus 40 (SV40) large T antigen (TAg), both free and bound to mature 70S and replicating 90S SV40 chromosomes, was prepared from lytically infected cells. The relative reactivity of the different TAg-containing fractions toward 10 monoclonal antibodies directed against three different regions in SV40 TAg and toward an antibody against the p53 protein was measured. The results for free TAg indicated that all of the determinants in both the amino-terminal (0.65 to 0.62 map units) and carboxy-terminal (0.28 to 0.17 map units) regions were highly reactive, whereas all five determinants located between 0.43 and 0.28 map units in the midregion of TAg were poorly reactive. For TAg bound to replicating chromosomes, all but one of the antibodies specific for TAg were highly reactive. Thus, antigenic sites in the middle of TAg, the region important for nucleotide binding and ATP hydrolysis (an activity required for viral DNA replication), were more accessible in TAg-replicating DNA complexes. As replicating molecules matured into 70S chromosomes, three or more determinants at different locations in TAg bound to chromatin became two- to fivefold less reactive, indicating other changes in TAg structure. Overall, at least nine different antigenic determinants in the TAg molecule were identified. Anti-p53 was reactive with about 10% of the free TAg and the same amount of SV40 chromosomes of all ages, suggesting that p53-TAg complexes are not preferentially associated with either replicating or mature viral chromosomes. When the reactivity of both mature and replicating labeled SV40 chromosomes with polyclonal tumor anti-T was measured as a function of time after purification, TAg bound to mature chromosomes appeared to dissociate about fourfold faster than that bound to replicating chromosomes. The relative amount of TAg in various subcellular fractions was measured by an enzyme-linked immunosorbent assay. Approximately 1.3% of the total TAg was estimated to be associated with SV40 chromosomes in infected cells. Based on the relative amounts of TAg and viral DNA in the 70S and 90S fractions, replicating chromosome-TAg complexes were estimated to bind 4.8 times more TAg per DNA molecule, on the average, than mature chromosome-TAg complexes. Together, these results are consistent with major differences in TAg structure when free and associated with replicating and nonreplicating SV40 chromosomes.
Topics: Animals; Antibodies, Monoclonal; Antigens, Viral, Tumor; Cell Line; DNA Replication; DNA, Viral; Epitopes; Genes, Viral; Haplorhini; Neoplasm Proteins; Nucleoproteins; Phosphoproteins; Simian virus 40; Tumor Suppressor Protein p53; Virus Replication
PubMed: 2422397
DOI: 10.1128/JVI.58.2.635-646.1986 -
Journal of Virology Sep 2004A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This...
A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This analysis included infectious isolates recovered between 1960 and 1999 from primary cultures of monkey kidney cells, from contaminated poliovaccines and an adenovirus seed stock, from human malignancies, and from transformed human cells. Maximum-parsimony and distance methods revealed distinct SV40 clades. However, no clear patterns of association between genotype and viral source were apparent. One clade (clade A) is derived from strain 776, the reference strain of SV40. Clade B contains isolates from poliovaccines (strains 777 and Baylor), from monkeys (strains N128, Rh911, and K661), and from human tumors (strains SVCPC and SVMEN). Thus, adaptation is not essential for SV40 survival in humans. The C terminus of the T-antigen (T-ag-C) gene contains the highest proportion of variable sites in the SV40 genome. An analysis based on just the T-ag-C region was highly congruent with the whole-genome analysis; hence, sequencing of just this one region is useful in strain identification. Analysis of an additional 16 strains for which only the T-ag-C gene was sequenced indicated that further SV40 genetic diversity is likely, resulting in a provisional clade (clade C) that currently contains strains associated with human tumors and human strain PML-1. Four other polymorphic regions in the genome were also identified. If these regions were analyzed in conjunction with the T-ag-C region, most of the phylogenetic signal could be captured without complete genome sequencing. This report represents the first whole-genome approach to establishing phylogenetic relatedness among different strains of SV40. It will be important in the future to develop a more complete catalog of SV40 variation in its natural monkey host, to determine if SV40 strains from different clades vary in biological or pathogenic properties, and to identify which SV40 strains are transmissible among humans.
Topics: Animals; Base Sequence; Cells, Cultured; Genetic Variation; Genome, Viral; Genomics; Haplorhini; Humans; Molecular Sequence Data; Neoplasms; Phylogeny; Polymorphism, Genetic; Sequence Analysis, DNA; Simian virus 40
PubMed: 15308725
DOI: 10.1128/JVI.78.17.9306-9316.2004 -
Journal of Virology Oct 1977Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting...
Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.
Topics: Cell Line; Chromosome Mapping; DNA Restriction Enzymes; DNA, Viral; Genetic Complementation Test; Helper Viruses; Mutation; Simian virus 40
PubMed: 198579
DOI: 10.1128/JVI.24.1.277-294.1977 -
Virology Feb 2009The story of SV40-induced tumorigenesis and cellular transformation is intimately entwined with the development of modern molecular biology. Because SV40 and other... (Review)
Review
The story of SV40-induced tumorigenesis and cellular transformation is intimately entwined with the development of modern molecular biology. Because SV40 and other viruses have small genomes and are relatively easy to manipulate in the laboratory, they offered tractable systems for molecular analysis. Thus, many of the early efforts to understand how eukaryotes replicate their DNA, regulate expression of their genes, and translate mRNA were focused on viral systems. The discovery that SV40 induces tumors in certain laboratory animals and transforms many types of cultured cells offered the first opportunity to explore the molecular basis for cancer. The goal of this article is to highlight some of the experiments that have led to our current view of SV40-induced transformation and to provide some context as to how they contributed to basic research in molecular biology and to our understanding of cancer.
Topics: Animals; Antigens, Polyomavirus Transforming; Cell Transformation, Viral; DNA, Viral; Mice; Simian virus 40; Virus Integration; Virus Replication
PubMed: 19070883
DOI: 10.1016/j.virol.2008.11.024 -
Cell Jun 1983
Review
Topics: Animals; Gene Expression Regulation; Operon; Simian virus 40; Transcription, Genetic
PubMed: 6305503
DOI: 10.1016/0092-8674(83)90410-5 -
Journal of Virology Jan 1981Late simian virus 40 (SV40) mRNA contains eight different cap structures which we have previously identified and mapped on the viral genome. As reported here, 5'-cap...
Late simian virus 40 (SV40) mRNA contains eight different cap structures which we have previously identified and mapped on the viral genome. As reported here, 5'-cap heterogeneity is a common feature to both the early and the late SV40 mRNA's. methyl-3H-labeled viral mRNA was purified from cells infected at 41 degrees C with SV40 mutant tsA209. Three different cap cores were identified: m7GpppGm, m7GpppCm, and m7GpppAm. An average of three to four m6A residues per mRNA molecule was found. RNase T2-resistant 32P-labeled early caps from tsA209-infected cells isolated and characterized. Six distinct cap I structures were identified: m7GpppCmpU (30%), m7GpppGmpC (24%), m7GpppAmpG (18%), m7GpppGmpU (13%), m7GpppGmpG (12%), and m7GpppAmpU (3%). A similar 5'-end heterogeneity was observed in early SV40 mRNA from BSC-1 cells infected with wild-type SV40 strain 777 in the presence of cytosine arabinoside and in the SV40 UV-transformed permissive line C-6. Five of these capped dinucleotides are complementary to DNA sequences at 0.66 map unit in a region previously identified by the primer extension method (Reddy et al., J. Virol. 30:279-296, 1979; Thompson et al., J. Virol. 31:437-438, 1979) as the 5' end of the early message. DNA sequences upstream from this region contain the TATTTAT (Hogness-Goldberg box), which is missing from upstream of the 5'-cap sites of late SV40 mRNA. Thus, 5'-end heterogeneity is not necessarily related to the presence or the absence of this putative transcriptional "initiation signal." When the possibility that SV40 5' caps represent transcriptional initiation sites is considered, the data also suggest that, on SV40 DNA, eucaryotic RNA polymerase II initiates transcription at multiple nucleotide sequences, including pyrimidines.
Topics: Animals; Base Sequence; Cell Transformation, Viral; Chlorocebus aethiops; Kidney; Methylation; Nucleic Acid Hybridization; RNA Caps; RNA, Messenger; RNA, Viral; Ribonucleases; Simian virus 40
PubMed: 6261002
DOI: 10.1128/JVI.37.1.7-16.1981 -
Proceedings of the National Academy of... Jul 1975We demonstrate here the usefulness of cytochalasin B enucleate cells for the study of the metabolism of cytoplasmic mRNA and for determining its half-life in animal...
We demonstrate here the usefulness of cytochalasin B enucleate cells for the study of the metabolism of cytoplasmic mRNA and for determining its half-life in animal cells. Simian virus 40 infected monkey cells in which the RNA had been labeled with [3H]uridine were enucleated, and the decay of the two prominent RNAs of simian virus 40, the 19S and 16S species, was measured by analysis on sucrose gradients. The results of these experiments, together with kinetic analysis of nuclear and cytoplasmic viral RNA, indicate a precursor-product relationship between the 19S and 16S cytoplasmic viral RNA species, which decay by first-order kinetics with a mean half-life of about 3 hr and 6 hr, respectively.
Topics: Animals; Cell Line; Cell Nucleus; Cytochalasin B; Haplorhini; Kinetics; RNA, Viral; Simian virus 40; Time Factors; Transcription, Genetic
PubMed: 170607
DOI: 10.1073/pnas.72.7.2587