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Journal of Virology Jan 1975Supercoiled BK papovavirus DNA was shown to transform hamster kidney cells using the calcium phosphate co-precipitation technique. The transformed cells contained...
Supercoiled BK papovavirus DNA was shown to transform hamster kidney cells using the calcium phosphate co-precipitation technique. The transformed cells contained intranuclear T-antigen(s) and rescuable virus and produced progressively growing tumors when inoculated into hamsters. A novel finding was the production in tumor-bearing animals of antinuclear antibody, which reacted against normal, untransformed cells; in addition, tumor serum contained antibody against virus-specific T-antigen(s).
Topics: Animals; Antibodies, Antinuclear; Antibodies, Neoplasm; Antigens, Neoplasm; Antigens, Viral; BK Virus; Calcium Phosphates; Cell Fusion; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Chemical Precipitation; Cricetinae; DNA, Viral; Kidney; Neoplasms, Experimental; Polyomavirus; Virus Replication
PubMed: 173887
DOI: 10.1128/JVI.17.1.247-253.1976 -
Proceedings of the National Academy of... Oct 1995We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA...
We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.
Topics: Animals; Base Sequence; Cyclic N-Oxides; DNA, Recombinant; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Carriers; Female; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Growth; Immunohistochemistry; Liposomes; Luciferases; Mice; Mice, Inbred BALB C; Mice, Transgenic; Molecular Sequence Data; Plasmids; Tissue Distribution
PubMed: 7568209
DOI: 10.1073/pnas.92.21.9742 -
Journal of Virology Oct 1991The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus... (Comparative Study)
Comparative Study
The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.
Topics: Amino Acid Sequence; Animals; Antigens, Polyomavirus Transforming; Cell Line; Cell Transformation, Neoplastic; Kinetics; Mice; Mice, Transgenic; Molecular Sequence Data; Papillomaviridae; Polyomaviridae; Protein Binding; Sequence Homology, Nucleic Acid; Tumor Suppressor Protein p53
PubMed: 1895390
DOI: 10.1128/JVI.65.10.5417-5424.1991 -
Journal of Virology Jan 1990The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the...
Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.
The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.
Topics: Animals; BK Virus; Blotting, Southern; Female; Gene Expression; Genes, Viral; Genetic Vectors; Herpes Simplex; Immunization; Mice; Mice, Inbred BALB C; Nucleic Acid Hybridization; Plasmids; Polyomavirus; Recombinant Proteins; Simplexvirus; Viral Envelope Proteins; Viral Structural Proteins
PubMed: 2152829
DOI: 10.1128/JVI.64.1.431-436.1990 -
Genes & Development Jun 1987One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the...
One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the level of appropriately initiated expression from the human heat shock protein 70 (hsp70) promoter. Sequences required for full activation lie upstream of the transcription initiation site and are distinct from sequences necessary for basal expression. These sequences also appear distinct from promoter sequences necessary for heat induction, serum induction, and induction by the papovavirus T antigens. The c-myc protein inhibits appropriately initiated expression from the mouse metallothionein I (MT-I) promoter. A mutation that removes 138 amino acids of exon 2 produces a c-myc gene product that is capable of activating the hsp70 promoter but is no longer capable of inhibiting MT-I expression, suggesting that these two properties reside in different domains of the c-myc protein. Expression from the adenovirus EII promoter is slightly inhibited, while expression from the SV40 early promoter is minimally affected by the c-myc protein. Both the spectrum of promoters regulated by the c-myc protein and the sequence requirements for that regulation differ from those of previously characterized viral trans-activating proteins. The data suggest that the c-myc protein can both stimulate and inhibit transcription from mammalian promoters in a novel manner.
Topics: Animals; Cells, Cultured; Gene Expression Regulation; Genes; Heat-Shock Proteins; Mice; Mice, Inbred BALB C; Plasmids; Promoter Regions, Genetic; Proto-Oncogenes; Transcription, Genetic; Transfection
PubMed: 3678827
DOI: 10.1101/gad.1.4.347 -
The Journal of Biological Chemistry Dec 2002RNA polymerase (pol) III transcription is abnormally active in fibroblasts transformed by polyomavirus (Py) or simian virus 40 (SV40). Several distinct mechanisms...
RNA polymerase (pol) III transcription is abnormally active in fibroblasts transformed by polyomavirus (Py) or simian virus 40 (SV40). Several distinct mechanisms contribute to this effect. In untransformed fibroblasts, the basal pol III transcription factor (TF) IIIB is repressed through association with the retinoblastoma protein RB; this restraint is overcome by large T antigens of Py and SV40. Furthermore, cells transformed by these papovaviruses overexpress the BDP1 subunit of TFIIIB, at both the protein and mRNA levels. Despite the overexpression of BDP1, the abundance of the other TFIIIB components is unperturbed following papovavirus transformation. In contrast, mRNAs encoding all five subunits of the basal factor TFIIIC2 are found at elevated levels in fibroblasts transformed by Py or SV40. Thus, both papovaviruses stimulate pol III transcription by boosting production of selected components of the basal machinery. Py differs from SV40 in encoding a highly oncogenic middle T antigen that localizes outside the nucleus and activates several signal transduction pathways. Middle T can serve as a potent activator of a pol III reporter in transfected cells. Several distinct mechanisms therefore contribute to the high levels of pol III transcription that accompany transformation by Py and SV40.
Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antigens, Polyomavirus Transforming; Base Sequence; Cell Division; Cell Transformation, Viral; DNA Primers; Mice; Molecular Sequence Data; RNA Polymerase III; RNA, Messenger; Simian virus 40; Transcription, Genetic
PubMed: 12370195
DOI: 10.1074/jbc.M201333200 -
Journal of Virology Dec 1979A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared...
A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared with a cloned wild-type virus (wt-501). Mutant pm-522 formed turbid plaques and grew more slowly than wt-501 in human embryonic kidney (HEK) cells. The immunofluorescence assay revealed that more HEK cells underwent abortive infection with pm-522 than with wt-501. Whereas wt-501 induced brain tumors and osteosarcomas, but no insulinomas, in hamsters, pm-522 induced brain tumors and insulinomas. The DNA of pm-522 was found by electrophoresis and electron microscopy to have a deletion (85 +/- 15 base pairs) and an insertion (40 +/- 10 base pairs) between map coordinates 0.708 and 0.725 from the endonuclease EcoRI cleavage site. These results demonstrate the presence of a viable deletion human papovarivus BK mutant capable of inducing insulinomas in hamsters.
Topics: Adenoma, Islet Cell; Animals; Antigens, Neoplasm; Antigens, Viral; BK Virus; Cricetinae; DNA, Viral; Genes, Viral; Humans; Mutation; Polyomavirus; Tumor Virus Infections
PubMed: 229273
DOI: 10.1128/JVI.32.3.934-942.1979 -
Journal of Virology Jun 1999Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian...
Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase alpha-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase alpha-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia coli as a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.
Topics: Antigens, Polyomavirus Transforming; DNA Helicases; DNA Replication; DNA-Binding Proteins; Humans; Replication Protein A; Simian virus 40; Trans-Activators; Viral Proteins; Virus Replication
PubMed: 10233951
DOI: 10.1128/JVI.73.6.4899-4907.1999 -
Molecular and Cellular Biology Dec 1991The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these...
The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these tumors develop focally after several months, it has been suggested that secondary cellular alterations are required to induce a tumor in this tissue. In contrast to SV40, the related lymphotropic papovavirus early region induces rapid nonfocal choroid plexus neoplasia in transgenic mice. Here, using hybrid gene constructs, we showed that T antigen from either virus in in fact sufficient to induce these tumors. Their abilities to induce proliferative abnormalities in other tissues, such as kidney and thymus, were also indistinguishable. Differences in the rate of choroid plexus tumorigenesis reflected differences in the control regions of the two viruses, rather than differences in T antigen per se. Under SV40 regulation, expression was limited to a fraction of the choroid plexus cells prior to the formation of focal tumors. When SV40 T antigen was placed under lymphotropic papovavirus control, in contrast, expression was generally uniform in the choroid plexus and rapid expansion of the tissue ensued. We found a direct relationship between T-antigen expression, morphological transformation, and proliferation of the choroid plexus epithelial cells. Analysis of mosaic transgenic mice indicated further that T antigen exerts its mitogenic effect cell autonomously. These studies form the foundation for elucidating the role of various T-antigen subactivities in tumorigenesis.
Topics: Animals; Antigens, Polyomavirus Transforming; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Choroid Plexus Neoplasms; Cloning, Molecular; Immunoenzyme Techniques; Kinetics; Mice; Mice, Transgenic; Papillomaviridae; Polyomaviridae; Simian virus 40
PubMed: 1658622
DOI: 10.1128/mcb.11.12.5968-5976.1991 -
Nucleic Acids Research Jul 1979We have determined the DNA sequences which correspond to the splicing regions in the transcripts of human papovavirus BKV, an evolutionary variant of SV40. To precisely... (Comparative Study)
Comparative Study
We have determined the DNA sequences which correspond to the splicing regions in the transcripts of human papovavirus BKV, an evolutionary variant of SV40. To precisely localize the excision points in the BKV sequence, we have conducted a preliminary analysis of numerous viral and eukaryotic splice site sequences. This analysis suggests that the preferred sequence for the donor site belongs to at least one of four groups: Pu↓GTAxG, Pu↓GTAxxT, Pu↓GTxxGT, Pu↓GTxAG (↓ = the cleavage site). These four groups derive from the two basic sequences, Pu↓GTxxG and Pu↓GTA. An optimal donor site might be: AG↓GTAAGT. The preferred sequence for the acceptor site is of the form PyPyxPyAG↓; no dinucleotide AG occurs within 13 nucleotides prior to the terminal AG of the 3' end of the intervening sequences. As we have found in this study of BKV splice sites, the sequences for the preferred donor and acceptor sites provide predictive value in localizing RNA splice points when the DNA sequence is known.
Topics: BK Virus; Base Sequence; DNA Restriction Enzymes; DNA, Viral; Genes, Viral; Humans; Polyomavirus; Simian virus 40; Species Specificity; Transcription, Genetic
PubMed: 225729
DOI: 10.1093/nar/6.10.3387