Polyoma „Tumor Antigen”: An Activator of Chromosome Replication?

Weil, Roger; Kára, J.

doi:10.1073/pnas.67.2.1011

Cited by 37 publications

(29 citation statements)

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“…Polyoma-induced chromosome replication, however, is accompanied by the production of progeny virus and is followed by cell lysis, but not by mitosis. The results are compatible with the idea that mouse cells lack a factor(s) required for the synthesis of SV40 viral progeny DNA and for the expression of late viral functions, and that for this reason infection with SV40 remains abortive (5,14,15).…”

Section: Discussionsupporting

confidence: 78%

“…Results from studies of the relationship between the transcription of SV40-specific viral RNA and T-antigen suggest that "early" SV40-specific messenger RNAt carries (the) information necessary for the production of T-antigen (manuscript in preparation). We tentatively represent the sequential events of the abortive infection by the simplified scheme: (a) transcription of "early" virus-specific ( (14). Polyoma-induced chromosome replication, however, is accompanied by the production of progeny virus and is followed by cell lysis, but not by mitosis.…”

Section: Discussionmentioning

confidence: 99%

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Analysis of the Events Leading to SV40-Induced Chromosome Replication and Mitosis in Primary Mouse Kidney Cell Cultures

May

Weil

1971

Proc. Natl. Acad. Sci. U.S.A.

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Abortive infection with simian virus 40 in confluent, "contact-inhibited", mouse kidney cell cultures was studied. The sequential events, in individual cells, are tentatively represented by the simplified scheme: Infection of confluent, "contact-inhibited" primary mouse kidney (MK) cell cultures with simian virus 40 (SV40) induces the replication of cellular DNA, while little, if any, viral DNA, capsid protein, or progeny virus is produced (1, 2). This abortive infection is followed, within a few weeks, by the appearance of foci of "transformed" cells (3,4).In this paper, evidence is presented that indicates that the abortive infection with SV40 actually triggers chromosome replication and mitosis. These results, and those presented elsewhere (5, and manuscript in preparation), show that this experimental system not only allows a detailed investigation of the early functions of SV40 and of their relation to the process of virus-induced cell "transformation", but that it is a promising model for the study of genetic regulation in mammalian cells. MATERIAL AND METHODSPrimary MK (6) cultures prepared from kidneys of 10-dayold white Swiss mice were grown in plastic Petri dishes (30-mm diameter, unless indicated otherwise) on glass coverslips (22 X 22 mm) (7). SV40 virus (large plaque, SVi strain) (8) and CV-1 cultures (an established monkey-kidney cell line) (9) were obtained in May 1969 from Dr. R. Cassingena. The virus used in our studies was grown and assayed for plaque-forming titer (PFU) on CV-1 cultures.Cultures were infected with SV40 virus by adding 0.15 ml of viral lysate and permitting adsorption to continue for 1 hr at 370C in a COrincubator. Unless indicated otherwise, the cultures were then covered with serum-free, reinforced Eagle's and Institut de Recherches medium (7) prewarmed to 370C. Essentially identical results were obtained when the cultures were washed after adsorption. The time at which virus was added to the cultures was considered to be time zero. Serial dilutions of the viral lysates were prepared in culture medium containing 2% calf serum (Microbiological Associates). In all experiments, control cultures were mock-infected and were treated subsequently in the same way as were the SV40-infected cultures.Immunofluorescence tests for SV40-specific T-antigen were performed according to the method of Fluorescein-conjugated anti-hamster globulin (from rabbits) was obtained from Progressive Laboratories, Inc., Baltimore, Md.Immunofluorescence tests for viral capsid antigen were performed as was described (7). The antiserum was obtained by repeated injection of highly purified SV40 capsids into the footpad of rabbits. The fluorescein-conjugated anti-rabbit globulin (from goats) was obtained from the Institut Pasteur (Paris). The relative number of cells with nuclear fluorescence for T-or capsid-antigen was determined in a Zeiss Ultraphot microscope. To obtain a value for each time interval a random sample of at least 1500 cells was counted.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Analysis of the Events Leading to SV40-Induced Chromosome Replication and Mitosis in Primary Mouse Kidney Cell Cultures

May

Weil

1971

Proc. Natl. Acad. Sci. U.S.A.

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“…The molecular and cytological events of the abortive infection in mouse kidney (MK) cells have been described (4,12,13). Under the conditions used in this study (10R PFU/ml; 370) a weak immunofluorescence reaction for T-antigen could be detected in 1-2% of the nuclei 7-8 hr after infection; later the relative number of positive nuclei (and the intensity of the immunofluorescence reaction) rapidly increased, reaching by 24 Under the conditions used (10P PFU/ml; 370) 1-5% of the nuclei contained T-antigen by [8][9] hr after infection; later the relative number of positive nuclei (and the intensity of the immunofluorescence reaction) rapidly increased, reaching 25-50% by 15 hr and 90-100% by [20][21][22][23][24] hr after infection. Time course of infection was similar in confluent BSC and Vero monkey kidney cell lines and also in confluent primary monkey kidney cell cultures.…”

Section: Resultsmentioning

confidence: 99%

“…However, in mixed incubation experiments, the molecular weight of T-antigen remains 86,000 not only in MK cell extracts, but also in the presence of extracts of other kinds of nonpermissive cells derived from the hamster. Earlier observations suggested that SV4O-(and polyoma-) induced host chromatin replication may be necessary but is not sufficient for viral DNA replication; hence it was postulated that permissive cells may contain a specific factor(s) absent in nonpermissive cells (4,23). Without proving it, our results are compatible with the hypothesis that this factor is a protease(s): 86 kDal T-antigen synthesized in nonpermissive cells would be able to induce a mitogenic response (leading to chromatin replication and mitosis), but it would allow autonomous replication of the viral DNA only if cleaved to the 82 kDal form.…”

Section: Identification Of the Polypeptides By Fingerprinting Analysismentioning

confidence: 99%

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Ahmad-Zadeh¹,

Allet²,

Greenblatt³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Simian-virus40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen (1, 4,1) of a part of SV40 DNA ("early region") that comprises about 45% of total length (5, 6). Evidence discussed elsewhere suggested that early 19S mRNA directs synthesis of a single, rather large protein, the SV40-specific T-antigen, which is required to induce lytic and abortive infection, to initiate and maintain cell transformation, and probably also to induce primary tumors in animals (details in refs. 4 and 7).In the present work we isolated T-antigen by immunoprecipitation. The T-antigen isolated in this way is shown to be coded by the viral DNA. We compared the molecular properties of T-antigen in abortive and lytic infection. Our The results were the same whether crude viral lysates or highly purified viral preparations were used for infection. Virus (0.3 ml per dish) was adsorbed at 370 for 90 min; the cultures were then covered with 10 ml of medium containing 10% bovine serum; in most experiments primary mouse kidney cultures were covered with serum-free medium (4).Parallel cultures were mock-infected with 0.3 ml of medium and then treated as SV40-infected cultures.Cultures were labeled with [asS]methionine Ci/ mmol; The Radiochemical Centre, Amersham, England) by addition of 3 ml per dish of prewarmed (370) medium (E serum) which contained only 2 glg/ml of unlabeled methionine (instead of 30 .ug) and 50 ,Ci/ml of [a5S]methionine. At the end of the labeling period the cultures were washed twice with 5 ml of cold (20) isotonic phosphate buffer; then the cells were gently scraped off in 1 ml of the same phosphate buffer with a silicone policeman and centrifuged for 5 min at 500 X g.Preparation of "Soluble Extracts." The pellets were suspended at concentrations varying between 107 and 108 cells in 5 ml of extraction buffer (2.5 mM Tris-HCl, pH 7.4, 0.5 M LiCl, 1 mM EDTA), sonicated (MSE 100 W Ultrasonic Disintegrator) four times for 15 sec in an ice-water bath, and then immediately centrifuged for 30 min at 30,000 X g at 40. The supernatant was removed and is referred to as "soluble extract." In early experiments phenylmethylsulfonyl fluoride (0.1-0.3 mg/ml) and dithiothreitol (1 mM) were present in the extraction buffer.Immunoprecipitation. Most experiments were performed with sera (anti-T) obtained from Syrian hamsters bearing tumors induced by inoculation of an SV40-transformed hamster cell line (9). One preparation was a gift of Drs. E. and P. May, others were made in our laboratory. Hamster anti-T sera (batches 3 X 1888 and 4 X 21) were received from the National Cancer Institute (Bethesda, Md.). The anti-T sera used had specific complement fixing titers of 160-640. Control serum was a pool of 15 normal adult Syrian hamster

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“…"Py-induced cellular DNA synthesis is neither preceded nor accompanied by an overall stimulation of cellular RNA synthesis nor is it followed by mitosis" (261) Py infection of highly confluent cells in culture has been frequently reported to stimulate an unscheduled round of cellular DNA synthesis (96,372,373). This synthesis, however, may occur out of normal cell cycle control, and it does not involve a full round of the cell cycle since mitosis does not follow the S phase (197,198).…”

Section: Dna Synthesismentioning

confidence: 99%

Natural Biology of Polyomavirus Middle T Antigen

Gottlieb

Villarreal

2001

Microbiol Mol Biol Rev

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“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

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Polyoma „Tumor Antigen”: An Activator of Chromosome Replication?

Cited by 37 publications

References 12 publications

Analysis of the Events Leading to SV40-Induced Chromosome Replication and Mitosis in Primary Mouse Kidney Cell Cultures

Analysis of the Events Leading to SV40-Induced Chromosome Replication and Mitosis in Primary Mouse Kidney Cell Cultures

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Natural Biology of Polyomavirus Middle T Antigen

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