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Journal of Virology Jun 1980Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose...
Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the "free" viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral "late" region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication.
Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Viral; DNA Restriction Enzymes; DNA, Viral; Molecular Weight; Polyomavirus; Rats; Recombination, Genetic
PubMed: 6247500
DOI: 10.1128/JVI.34.3.615-626.1980 -
Journal of Virology Jan 1983The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with...
The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.
Topics: Amino Acid Sequence; Antigens, Viral; Antigens, Viral, Tumor; Base Sequence; DNA Replication; DNA Restriction Enzymes; Genes, Viral; JC Virus; Polyomavirus; RNA Splicing; RNA, Viral; Virus Replication
PubMed: 6296460
DOI: 10.1128/JVI.45.1.73-79.1983 -
Microbiology and Molecular Biology... Jun 2001"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The... (Review)
Review
"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.
Topics: Animals; Antigens, Polyomavirus Transforming; Apoptosis; Cell Differentiation; Cell Transformation, Viral; Eukaryotic Cells; Mice; Mice, Knockout; Polyomavirus; Signal Transduction; Transcription, Genetic; Virus Replication
PubMed: 11381103
DOI: 10.1128/MMBR.65.2.288-318.2001 -
Proceedings of the National Academy of... Dec 1985Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region,...
Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp60c-src and a new protein of approximately equal to 61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of approximately equal to 72,000 Da.
Topics: Animals; Antigens, Viral, Tumor; Immunosorbent Techniques; Macromolecular Substances; Mice; Molecular Weight; Polyomavirus; Protein Binding; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins pp60(c-src); Viral Proteins
PubMed: 2415976
DOI: 10.1073/pnas.82.23.7952 -
The EMBO Journal Mar 1984We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the...
We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the existence of the complex using both monoclonal antibodies specific for middle-T and antibodies raised against synthetic peptides corresponding to sequences from both middle-T and pp60c-src. The complex was retained during partial purification of middle-T and was stable to incubation under various conditions. A survey of a number of mutants of middle-T antigen showed that there was a complete correlation between the ability of middle-T to accept phosphate in the in vitro kinase reaction and the presence of a middle-T: pp60c-src complex. This result is in accord with our hypothesis that middle-T itself is not a protein kinase but rather that pp60c-src phosphorylates middle-T. All mutant forms of middle-T antigen capable of transformation had associated pp60c-src. The middle-T of two non-transforming mutants (hr-t mutants) did not have associated pp60c-src, whereas other non-transforming middle-T species did associate with pp60c-src. We propose that the complex plays an essential role in transformation by polyoma virus, but that the existence of the complex per se may not be sufficient.
Topics: Antigens, Viral, Tumor; Cell Transformation, Viral; Cells, Cultured; Macromolecular Substances; Oncogene Protein pp60(v-src); Polyomavirus; Protein Kinases; Viral Proteins
PubMed: 6325177
DOI: 10.1002/j.1460-2075.1984.tb01852.x -
Journal of Virology Mar 1979At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T...
At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.
Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cricetinae; Methionine; Mice; Molecular Weight; Peptides; Polyomavirus; Viral Proteins
PubMed: 221679
DOI: 10.1128/JVI.29.3.881-887.1979 -
Journal of Virology Apr 1975The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in...
The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.
Topics: Carbon Radioisotopes; Centrifugation, Zonal; Electrophoresis, Polyacrylamide Gel; Iodine; Lysine; Molecular Weight; Papillomaviridae; Peptides; Polyomaviridae; Polyomavirus; Protein Hydrolysates; Simian virus 40; Tritium; Trypsin
PubMed: 163921
DOI: 10.1128/JVI.15.4.828-835.1975 -
Initiation of polyoma virus DNA replication in vitro and its dependence on the viral gene A protein.Nucleic Acids Research Oct 1980Initiation of polyoma virus DNA replication is dependent on the activity of the early protein affected by the tsa mutations (large-T antigen). An in vitro DNA...
Initiation of polyoma virus DNA replication is dependent on the activity of the early protein affected by the tsa mutations (large-T antigen). An in vitro DNA synthesizing system blocked at the initiation stage was designed by preparing nuclei from cells shifted to high temperature after infection with a polyoma tsa mutant. Addition to these nuclei of extracts from wild type virus-infected cells resulted in a limited, but reproducible stimulation of deoxynucleoside monophosphate incorporation. At least for a significant part, this stimulation was shown to correspond to an increased synthesis of molecules identified as polyoma replicative intermediates by their sedimentation coefficient and endonuclease Hpa II cleavage pattern. The non-random distribution of label observed among restriction fragments was that expected from an initiation event occuring at the physiological origin. This activity was reduced to background level in extracts from tsa-infected cells shifted to high temperature and was specifically inhibited by addition of Fab fragments from anti-polyoma virus T antigen immunoglobulins.
Topics: Animals; Antigens, Viral; Cell Line; Cell Nucleus; DNA Replication; DNA Restriction Enzymes; DNA, Circular; DNA, Viral; Genes, Viral; Kinetics; Mice; Molecular Weight; Mutation; Polyomavirus; Viral Proteins; Virus Replication
PubMed: 6253915
DOI: 10.1093/nar/8.19.4377 -
Journal of Virology Mar 1977A two-step hybridization with polyoma DNA was used to study the composition of giant RNA molecules synthesized in mouse kidney cells late in productive infection by...
A two-step hybridization with polyoma DNA was used to study the composition of giant RNA molecules synthesized in mouse kidney cells late in productive infection by polyoma virus. Giant molecules longer than a complete transcript of the polyoma genome were purified from cells that had been pulse-labeled for 30 min with [3H]uridine and annealed, under mild conditions (50% formamide, 37 degrees C), with polyoma DNA loaded on nitrocellulose filters. Hybridized RNA (6 to 7% of the entire population of 3H-labeled molecules and up to 15% of the molecules containing polyadenylic acid [poly(A)]] was eluted and annealed a second time with polyoma DNA under more stringent conditions. In this second step, 75% of the 3H-labeled RNA formed an RNase-resistant hybrid. Under the same conditions, complementary RNA hybridized with polyoma DNA to a maximal extent of 80%. Since the difference between 75 and 80% is within the experimental error of the hybridization assay, it is inferred that the giant molecules selected by the first hybridization may consist entirely of virus-specific sequences or contain, at the most, a minor fraction of nonviral sequences. To examine the possibility that such nonviral sequences are clustered at the 3'-terminus of these molecules, poly(A)+ giant RNA, which had not been preselected by hybridization with polyoma DNA, was fragmented by a limited alkaline hydrolysis. Fragments linked to the poly(A) segment were separated from the rest of the cleavage products. A one-step hybridization with polyoma DNA revealed that both fractions contain 8 to 10% of virus-specific sequences. These results indicate that the 3'-termini of the poly(A)+ polyoma-specific giant RNA molecules consist of viral rather than nonviral sequences.
Topics: Animals; Base Sequence; Cell Line; DNA, Viral; Mice; Mice, Inbred C3H; Nucleic Acid Hybridization; Poly A; Polyomavirus; RNA; RNA, Viral
PubMed: 191649
DOI: 10.1128/JVI.21.3.831-842.1977 -
Proceedings of the National Academy of... Oct 2005The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent...
The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.
Topics: Actins; Animals; Biological Transport; Cell Membrane; Cholesterol; Diffusion; Fibroblasts; Gangliosides; Lipid Bilayers; Membrane Lipids; Mice; Microscopy, Fluorescence; Motion; Polyomavirus; Protein-Tyrosine Kinases
PubMed: 16219700
DOI: 10.1073/pnas.0504407102