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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 -
Journal of Virology Sep 1972Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain...
Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain the same number of copies of polyoma virus deoxyribonucleic acid (DNA) per cell (eight) as revertants with a subdiploid or a subtetraploid chromosome number. The results indicate that the duplication of chromosomes in the subtetraploid revertants did not include the chromosomes that carry the viral genome. The virus DNA in both transformed and revertant cells was associated with high-molecular-weight cell DNA. Reversion of the properties of transformed cells was, therefore, not associated either with a decrease in number of virus DNA copies per cell or with a lack of association of the virus DNA with cell DNA.
Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation, Density Gradient; Cricetinae; DNA; DNA, Neoplasm; DNA, Viral; Diploidy; Genetic Variation; Nucleic Acid Hybridization; Phosphorus Isotopes; Polyomavirus; Polyploidy; RNA, Viral; Tritium
PubMed: 4342053
DOI: 10.1128/JVI.10.3.456-461.1972 -
Journal of Virology Oct 1982We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The...
We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.
Topics: Animals; Antigens, Viral; Antigens, Viral, Tumor; Base Sequence; Cell Line; Genes, Viral; Mice; Nucleic Acid Heteroduplexes; Nucleic Acid Hybridization; Polyomavirus; RNA Splicing; RNA, Messenger; RNA, Viral; Time Factors; Transcription, Genetic; Viral Proteins
PubMed: 6292484
DOI: 10.1128/JVI.44.1.175-188.1982 -
Nucleic Acids Research Aug 1987In marked contrast to simian virus 40 (SV40), polyoma virus (PyV) has been reported to replicate discontinuously on both arms of replication forks. In an effort to...
In marked contrast to simian virus 40 (SV40), polyoma virus (PyV) has been reported to replicate discontinuously on both arms of replication forks. In an effort to clarify the relationship between the mechanisms of DNA replication in these closely related viruses, the distribution of RNA-primed DNA chains at replication forks was examined concurrently in PyV and SV40 replicating DNA purified from virus-infected cells. About one third of PyV DNA chains contained 7 to 9 ribonucleotides covalently linked to their 5'-end. A similar fraction of DNA chains from replicating SV40 DNA contained an oligoribonucleotide that was 6 to 9 residues long and began with either (p)ppA or (p)ppG. Greater than 80% of PyV or SV40 RNA-primed DNA chains hybridized specifically to the retrograde template. Moreover, at least 95% of the RNA-primed DNA chains from either PyV or SV40 whose initiation sites could be mapped to unique nucleotide locations originated from the retrograde template. Therefore, PyV and SV40 DNA replication forks are essentially the same; DNA synthesis is discontinuous predominantly, if not exclusively, on the retrograde template.
Topics: DNA Replication; DNA, Viral; Models, Genetic; Polyomavirus; RNA; RNA, Viral; Replicon; Templates, Genetic; Virus Replication
PubMed: 2442727
DOI: 10.1093/nar/15.16.6369 -
Proceedings of the National Academy of... Dec 1978Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and...
Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.
Topics: Animals; Antigens, Neoplasm; Antigens, Viral; DNA, Viral; Female; Molecular Weight; Oocytes; Polyomavirus; Protein Biosynthesis; Simian virus 40; Transcription, Genetic; Viral Proteins; Xenopus
PubMed: 216012
DOI: 10.1073/pnas.75.12.6073 -
The EMBO Journal 1982Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are...
Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.
Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Viral; DNA Restriction Enzymes; DNA, Circular; Female; Mice; Mice, Inbred Strains; Mutation; Placenta; Polyomavirus; Pregnancy; Species Specificity; Teratoma; Trophoblasts
PubMed: 6327274
DOI: 10.1002/j.1460-2075.1982.tb01349.x -
Molecular and Cellular Biology Apr 1983To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) gene early after transfer into TK-deficient mouse...
To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) gene early after transfer into TK-deficient mouse cells and the subsequent development of stable TK-positive transformants, we constructed a series of recombinant plasmids containing the herpes simplex virus TK gene joined with various segments of the polyoma virus genome and microinjected them into the nuclei or cytoplasm of LTK-A cells (TK(-), APRT(-)). The frequency of nucleus-injected cells expressing TK after 1 day, measured by autoradiography of cells incubated with [(3)H]thymidine, increased approximately 30-fold when the plasmids contained the polyoma virus origin of replication. The origin includes sequences with homology to the simian virus 40 origin of replication and adjoining sequences, including a recently defined transcription-enhancing sequence. After microinjection of a single origin-containing plasmid molecule per cell, TK expression was detected in approximately 50% of the injected cells. When a larger number of origin-containing plasmid molecules were injected per cell, all cells showed early TK activity. When the entire polyoma virus early region was present, neighboring uninjected cells became TK positive. When plasmids were injected into the cell cytoplasm, approximately 400 times as many molecules per cell were needed to cause early TK activity. The frequency of stable transformation observed 2 weeks after nuclear injection of 10 to 20 polyoma virus origin-containing plasmid molecules per cell was at least 2 orders of magnitude greater than with plasmids containing the TK gene alone. The greatest enhancement of stable TK transformation was obtained with plasmids containing the origin alone, when the maximum frequency of stable transformation was 5%. The addition of the coding regions for the small and medium T antigens or the entire early region significantly decreased TK transformation frequency in a copy-dependent fashion. The timing of stabilization of TK-positive transformation was analyzed by releasing hypoxanthine-aminopterin-thymidine selection pressure at various times after microinjection, culturing the cells in nonselective medium, and assaying for TK activity. Stabilization was found to occur between 3 and 6 days after nuclear injection. Cells injected with a plasmid containing the origin and the early region were examined for expression of the large T antigen with polyoma virus antitumor serum and immunofluorescent staining. The expression of the large T antigen was clearly associated with a cytopathic effect. TK-positive clones observed 2 weeks after injection of the plasmid were uniformly T antigen negative. Cytotoxicity may be the result of plasmid replication and toxic levels of T antigen or TK. In addition, expression of the large T antigen may block stabilization by preventing the integration of origin-containing plasmid molecules.
Topics: Animals; Antigens, Viral; Antigens, Viral, Tumor; Cells, Cultured; Gene Expression Regulation; Mice; Phenotype; Plasmids; Polyomavirus; Simplexvirus; Thymidine Kinase; Time Factors; Transformation, Genetic
PubMed: 6304496
DOI: 10.1128/mcb.3.4.511-522.1983 -
Antimicrobial Agents and Chemotherapy Jan 1977This report describes the inactivation of lipid-containing viruses by several long-chain alcohols. A striking peak in antiviral activity was found for saturated alcohols...
This report describes the inactivation of lipid-containing viruses by several long-chain alcohols. A striking peak in antiviral activity was found for saturated alcohols having chain lengths from 10 to 14 carbons. Viruses having different membrane structure showed different susceptibilities to alcohols having different chain lengths and structural features. Decanol, dodecanol, and tetradecanol readily inactivated herpes simplex virus and the enveloped bacterial virus phi6. The lipid-containing virus PM2 was susceptible to decanol and dodecanol but comparatively unsusceptible to tetradecanol. The branched-chain alcohol phytol, a naturally occurring component of chlorophyll, was active against phi6 and herpes simplex virus but not against PM2. Polyoma virus and the bacteriophage phi23-1-a, which do not contain lipids, were not susceptible to inactivation by any of the alcohols tested. Experiments were also carried out to determine the effects of these compounds on cells. At 0.5 mM, decanol lysed human embryonic lung cells, erythrocytes, and the bacterial hosts for phi6 and PM2. Dodecanol, tetradecanol, and phytol at this concentration were less damaging to cells. At 0.05 mM, none of the alcohols caused observable cytopathic effects on human embryonic lung cells, although several of the alcohols at this concentration were active against herpes simplex virus. Our findings suggest that dodecanol, tetradecanol, and phytol may warrant further studies as potential antiviral agents, particularly for topical application to virus-infected areas of the skin.
Topics: Antiviral Agents; Bacteriophages; Fatty Alcohols; Humans; Phytol; Polyomavirus; Simplexvirus; Viruses
PubMed: 189684
DOI: 10.1128/AAC.11.1.98 -
Journal of Virology Jul 1980A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of...
A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.
Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Base Sequence; Cell Line; Cell Transformation, Viral; Chromosome Mapping; Cricetinae; Electrophoresis, Polyacrylamide Gel; Genes, Viral; Mice; Mutation; Peptide Fragments; Phenotype; Polyomavirus; Rats; Temperature
PubMed: 6251270
DOI: 10.1128/JVI.35.1.219-232.1980 -
Proceedings of the National Academy of... Dec 1998In an effort to understand the unusual cytogenetic damage earlier encountered in the Yanomama Indians, plasma samples from 425 Amerindians representing 14 tribes have...
The JC and BK human polyoma viruses appear to be recent introductions to some South American Indian tribes: there is no serological evidence of cross-reactivity with the simian polyoma virus SV40.
In an effort to understand the unusual cytogenetic damage earlier encountered in the Yanomama Indians, plasma samples from 425 Amerindians representing 14 tribes have been tested for hemagglutination inhibition antibodies to the human JC polyoma virus and from 369 Amerinds from 13 tribes for hemagglutination inhibition antibodies to the human BK polyoma virus. There is for both viruses highly significant heterogeneity between tribes for the prevalence of serum antibody titers >/=1/40, the pattern of infection suggesting that these two viruses only relatively recently have been introduced into some of these tribes. Some of these samples, from populations with no known exposure to the simian polyoma virus SV40, also were tested for antibodies to this virus by using an immunospot assay. In contrast to the findings of Brown et al. (Brown, P., Tsai, T. & Gajdusek, D. C. (1975) Am. J. Epidemiol. 102, 331-340), none of the samples was found to possess antibodies to SV40. In addition, no significant titers to SV40 were found in a sample of 97 Japanese adults, many of whom had been found to exhibit elevated titers to the JC and BK viruses. This study thus suggests that these human sera contain significant antibody titers to the human polyoma viruses JC and BK but do not appear to contain either cross-reactive antibodies to SV40 or primary antibodies resulting from SV40 infection.
Topics: Adult; Antibodies, Viral; BK Virus; Central America; Cross Reactions; Ethnicity; Hemagglutination Inhibition Tests; Humans; Indians, Central American; Indians, South American; JC Virus; Japan; Simian virus 40
PubMed: 9861002
DOI: 10.1073/pnas.95.26.15525