Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40

Fluck, Michele M.; Benjamin, Thomas L.

doi:10.1016/0042-6822(79)90185-5

Virology

1979

DOI: 10.1016/0042-6822(79)90185-5

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Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40

Michele M. Fluck

Thomas L. Benjamin

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1979

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Cited by 114 publications

(70 citation statements)

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“…Characterization of cell lines derived from Flll cells. The transformation activity of the various DNA tumor virus oncogenes was investigated by analyzing cell lines derived after retrovirus infection of rat Flll cells (13). These cells are highly contact inhibited and have been extensively used to study transformation by papovaviruses.…”

mentioning

confidence: 99%

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Jat¹,

Cepko²,

Mulligan³

et al. 1986

Mol. Cell. Biol.

169

112

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We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat Flll cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorageindependent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.The DNA tumor viruses polyomavirus (Py) and simian virus 40 (SV40) are both capable of transforming both primary and established cell lines in vitro (for a review see reference 70). Extensive work has shown that genes encoded by the early regions of these viruses are both necessary and sufficient for transformation (1,10,12,14,15,18,29,38,43,49,67; for a review see reference 70). Analysis of the respective roles of these genes has been complicated because the early regions are multiply spliced to specify more than one protein: Py encodes three antigens, the large T (100 kilodaltons [kDa]), the middle T (56 kDa), and the small T (22 kDa) (20,21,24,25,58); SV40 encodes two antigens, the large T (95 kDa) and the small T (20 kDa) (48,51,69). In addition, transformation assays with DNA tumor viruses have generally involved infection or transfection of a cell population and selection of a small fraction of cells with a particular phenotype. For example, this selection could be based on either focus formation, anchorage-independent growth, or growth in low serum, and thus, this type of assay does not necessarily reveal the average phenotype resulting from interaction of the oncogene with the cell. Moreover, the process by which the viral DNA segment integrates into cellular chromosomes can give rise to multiple copies and rearrangements. The resulting variation in levels of viral mRNA and gene expression can significantly affect the range of phenotypes of the transformants. Previous studies of the variation in phenotype observed after transformation by SV40 detected clonal cell lines that ranged over completely, partially, or minimally transformed (50, 56).We therefore undertook a systematic analysis of the role of the various genes encoded by the early regions of these viruses by isolating cell lines which constitutively express these proteins without prior selection ...

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mentioning

confidence: 99%

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Jat¹,

Cepko²,

Mulligan³

et al. 1986

Mol. Cell. Biol.

169

112

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“…Nevertheless, several lines of evidence indicate that the small T antigen alone is insufficient to cause transformation and that an intact large T antigen, although required for some initial event during transformation, is not required for continuous expression of the transformed phenotype (7,10). Therefore, attention has focused recently on the middle T antigen as the viral protein potentially most directly involved in altered cell growth control (11).…”

mentioning

confidence: 99%

“…The temperature-sensitive tsA mutations, which affect the large T antigen, block the ability of the mutant virus to transform at the nonpermissive temperature (3,4). The host range nontransforming (hr-t) mutations, which affect the small and middle T antigens coordinately, render the mutant virus unable to transform infected cells (5)(6)(7).…”

mentioning

confidence: 99%

Antibodies specific for the polyoma virus middle-size tumor antigen.

Walter¹,

Hutchinson²,

Hunter³

et al. 1981

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

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We have obtained antibodies specific for the polyoma virus middle-size tumor antigen (middle T antigen) by immunizing rabbits with a synthetic peptide, Lys-Arg-Ser-ArgHis-Phe, corresponding to the six carboxy-terminal amino acids of the middle T antigen predicted from the nucleotide sequence ofpolyoma DNA. The antipeptide serum precipitates the polyoma middle T antigen but not the small or large tumor antigens, and precipitation is inhibited in the presence of the peptide. Two cellular proteins, 30,000 and 26,000 daltons, are also precipitated specifically by the antipeptide serum and may have amino acid sequences related to the peptide. Two other cellular proteins, 33,000 and 25,000 daltons, are precipitated only in the presence of the peptide and may associate with it in cell extracts. Antisera directed against synthetic peptides are likely to be important in various ways, including the production of antibodies directed against particular determinants and the recognition of unknown proteins whose genes have been analyzed.The early region of the polyoma virus genome encodes three proteins, called the large, middle, and small tumor antigens (T antigens) with Mrs of approximately 89,000, 48,000, and 22,000, respectively (1, 2). The coding regions for the three proteins overlap: the three proteins have common NH2-terminal regions encoded between 74 and 79 map units, and the small and middle T antigens have common sequences encoded between 79 and 85 map units that are not present in the large T antigen. The large and middle T antigens have unique sequences encoded between 86 and 99 map units, translated in different reading frames from the viral DNA (1, 2). In addition, the large T antigen has unique sequences encoded between 99 and 26 map units.Mutations affecting the T antigens suggest that the T antigens are involved in cell transformation. The temperature-sensitive tsA mutations, which affect the large T antigen, block the ability of the mutant virus to transform at the nonpermissive temperature (3, 4). The host range nontransforming (hr-t) mutations, which affect the small and middle T antigens coordinately, render the mutant virus unable to transform infected cells (5-7).Viable deletion mutations have been located in the common coding region for the large and middle T antigens between 86 and 99 map units. These mutations in some cases affect the frequency and expression of transformation (8,9).

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“…The genomic organization of this -2,500-base-pair region is such that the most 5' coding segments of each gene are identical, and the remaining 3' regions are unique. Specifically, T and t have a common, N-terminal 82-amino-acid sequence, and their respective C-terminal 626 and 92 residues are unique.Both proteins can function in a complementary fashion in promoting the maintenance of a neoplastic phenotype in certain established rodent cell lines which have been infected under selected conditions (3,4,11,13,19,28,30,31,34). Hence, it might be suspected that their unique coding segments are important to their respective roles in this process.…”

mentioning

confidence: 99%

“…Both proteins can function in a complementary fashion in promoting the maintenance of a neoplastic phenotype in certain established rodent cell lines which have been infected under selected conditions (3,4,11,13,19,28,30,31,34). Hence, it might be suspected that their unique coding segments are important to their respective roles in this process.…”

mentioning

confidence: 99%

The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.

Bikel¹,

Mamon²,

Brown³

et al. 1986

Mol. Cell. Biol.

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The smail t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function.The transforming region of simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen (T and t, respectively) (25). The genomic organization of this -2,500-base-pair region is such that the most 5' coding segments of each gene are identical, and the remaining 3' regions are unique. Specifically, T and t have a common, N-terminal 82-amino-acid sequence, and their respective C-terminal 626 and 92 residues are unique.Both proteins can function in a complementary fashion in promoting the maintenance of a neoplastic phenotype in certain established rodent cell lines which have been infected under selected conditions (3,4,11,13,19,28,30,31,34). Hence, it might be suspected that their unique coding segments are important to their respective roles in this process. By contrast, T alone can sustain the neoplastic phenotype of other SV40 transformants, whereas t alone appears to be ineffective in this regard (6,7,26,31). Given these findings, it is possible that, in some settings, T can perform its own function and that of t or that certain cells can supply a t-like function.Little is known of how t or T operates in the viral transforming mechanism, and there is limited information on the in vitro functional properties of t. By immunofluorescent and biochemical extraction analysis, both the SV40 and the polyomavirus t protein appear to be located in both the nucleus and the cytoplasm of infected cells (10,22,40). When introduced into rat cells, the SV40 protein can promote the dissolution of the actin cytoskeleton (2, 14, 15, 26). Moreover, it can render certain cells relatively resistant to the DNA synthesis inhibition effect of theophylline, stimulate them to synthesize a new centriolar antigen(s), and, together with T, promote persistent cycling of Gl-arrested cells (5,15,27,29,33). Whether any of these properties contributes to its transformation maintenance function is not yet clear, but this can eventually be tested by genetic means.In this regard, the technology of synthetic oligonucleotide...

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Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40

Cited by 114 publications

References 71 publications

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Antibodies specific for the polyoma virus middle-size tumor antigen.

The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.

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