Interaction of partially purified simian virus 40 T antigen with circular viral DNA molecules.

Simian virus 40 largetumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a, broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.Simian virus 40 (SV40) large tumor antigen (T Ag) is encoded by the viral A locus (1, 2), which governs initiation of DNA synthesis and the establishment of viral transformation (3, 4). Found in the nuclei of cells infected (5) and transformed (6) by SV40, T Ag is a DNA-binding protein (7-11) that binds DNA with heterogeneous affinities (7,12). Mutation in the viral A locus has been shown to decrease the DNA-binding affinity of T Ag (13,14). T Ag also sediments heterogeneously with sedimentation coefficients from 5S to greater than 22S (7,(15)(16)(17)(18)(19)(20). Mutation of the A locus causes T Ag to sediment as a 5-6S species (17, 18), corresponding to the 94,000 dalton monomer (13). Furthermore, T Ag, a phosphoprotein (21), is heterogeneously phosphorylated (22), generating multiple isoelectric focusing species with differing contents of phosphate. Differential phosphorylation may play a physiological role in generating the heterogeneous aggregating and DNA-binding forms of T Ag. Similarly, the addition and removal of the phosphate groups of acidic chromatin-associated proteins of eukaryotic cells has been suggested as a control mechanism for the regulation of their DNA-binding properties (23). In addition, differential phosphorylation states can affect the binding affinities of subunits for each other, as in glycogen phosphorylase (24).Two recently discovered host gene products, the Mr 48,000 and 55,000 host tumor antigens (HTA), also termed nonviral T Ags, have been shown to complex with T Ag in vitro (19,20,25,26 McCormick and Harlow (20) have proposed that the "53K NVT"-associated form oflarge T Ag becomes more highly phosphorylated than the free form. The 48,000 and 55,000 HTAs have been shown to be unrelated immunologically and structurally (19). The broad physiological significance of these nuclear phosphoproteins has been established by the fact that they are induced in mouse, rat, hamster, and human cells transformed by SV40 (19). They are expressed, in the absence of SV40, in cells transformed by a variety ofcarcinogenic agentsincluding the RNA tumor virus, murine sarcoma virus, and the chemical carcinogen methyicholanthrene (27)-and in spontaneously transformed fibro...

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confidence: 99%

Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen.

Greenspan¹,

Carroll²

1981

“…The T-antigen has so far been characterized primarily immunologically, employing sera from animals bearing SV40-induced tumors (11-13). T-antigen has been partially purified (14-16) and is now considered to be a DNA-binding protein (17)(18)(19)(20) with the molecular weight of the denatured polypeptide estimated to be from 70,000 (21) to 100,000 (22).Comparison of the properties of the T-antigen after infection with either wild-type or A-mutant virus at the restrictive temperature has supplied evidence that this antigen (or a part thereof) is encoded by viral DNA (22-24). To demonstrate directly that the information for the SV40 T-antigen is encoded in the viral genome, and to study its expression further, we have investigated the cell-free translation of early SV40 mRNA.…”

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confidence: 99%

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Prives

Gilboa

Revel

et al. 1977

153

133

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It Complementation studies with temperature-sensitive mutants of the tumor-causing simian virus 40 (SV40) indicate that it may have as few as three primary gene products (1). Only one of these, the A gene product, has been directly linked to the process of virus-induced neoplastic transformation (2-5). Cells in the early phase of productive lytic infection and virus-transformed cells exhibit some common features. In each case, a species of virus-specific RNA is induced, which hybridizes to the same strand of SV40 DNA (6, 7) and to the same fragments of SV40 DNA generated by restriction endonucleases (8, 9). At least three virus-associated antigens, T, U, and TSTA, have been detected both early in lytic infection and in transformed cells (10). The T-antigen has so far been characterized primarily immunologically, employing sera from animals bearing SV40-induced tumors (11-13). T-antigen has been partially purified (14-16) and is now considered to be a DNA-binding protein (17)(18)(19)(20) with the molecular weight of the denatured polypeptide estimated to be from 70,000 (21) to 100,000 (22).Comparison of the properties of the T-antigen after infection with either wild-type or A-mutant virus at the restrictive temperature has supplied evidence that this antigen (or a part thereof) is encoded by viral DNA (22-24). To demonstrate directly that the information for the SV40 T-antigen is encoded in the viral genome, and to study its expression further, we have investigated the cell-free translation of early SV40 mRNA.We report that SV40 early mRNA from infected cells, and SV40 cRNA transcripts made in vitro, direct the synthesis in the wheat-germ cell-free system of polypeptides that can be specifically immunoprecipitated by antiserum to SV40 Tantigen. The products directed by "in vivo" mRNA and synthetic cRNA differ, however, in their electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide gels. The former products correspond to the T-antigen immunoprecipitated from extracts of infected cells and the largest of these has an estimated molecular weight of 90,000. A preliminary description of these findings has been given previously (25). Antiserum to SV40 T-antigen was obtained from Flow Laboratories. Goat anti-hamster serum was a gift from M. Fogel. MATERIALS AND METHODSCells and Viruses. Plaque-purified SV40 (strain 777) was grown in the BS-C-1 line of African green monkey cells as previously described (26).Cell Extracts. Cultures (107 cells) of uninfected or SV40-infected cells (multiplicity of infection = 2 X 109 plaqueforming units/ml) were labeled from 47 to 48 hr post infection with [35S]methionine, (20 MCi/ml of methionine-free medium). After...

“…Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei of SV80 cells, a con- (21,22). Historically, purification of T antigen has been hampered by various difficulties.…”

Section: Introductionmentioning

confidence: 99%

“…Thus far, in vitro T antigen studies have been carried out in our laboratory with partially purified protein (21,22). Historically, purification of T antigen has been hampered by various difficulties.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.

Tenen¹,

Garewal²,

Haines³

et al. 1977

Self Cite

Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei of SV80 cells, a con- (21,22). Historically, purification of T antigen has been hampered by various difficulties. These have included instability of the complement-fixing activity of the antigen (23), its relatively low concentration in some SV40-infected permissive and transformed cell lines (10, 24), and low yields after multiple chromatographic procedures (10, 24). In this report, we describe the isolation of extensively purified T antigen in microgram quantities. The antigen was extracted from a cell line, SV80, shown previously to contain relatively high concentrations of the protein (24). A five-step purification procedure was used. The sonicated suspension was centrifuged in a JA-14 rotor for 15 min at 15,000 rpm at 40, and the supernatant was retained. It was fractionated between 30 and 60% (NH4)2SO4 saturation, and the final pellet was redissolved in sufficient buffer C (0.05 M Tris-HCI, pH 7.8/10% glycerol/3 mM DTT/0.02% Triton X-100/PMSF 100 jg/ml/TPCK, 50 jig/ ml/TLCK, 20 Ag/ml) to dissolve all of the protein (usually 30-50 ml for material from a 60-g cell pack). This fraction was dialyzed for 6 hr against two changes of 40 volumes of buffer C, and any insoluble protein was removed by centrifugation. It was loaded on a column of DEAE-cellulose (DE-52, Whatman) (120 ml packed volume per 60 g of cells) equilibrated in buffer C. After washing with sufficient buffer C to bring the A2o of the effluent to base-line levels (approximately 2 column Abbreviations: SV40, simian virus 40; T antigen, simian virus 40 tumor antigen; NaDodSO4, sodium dodecyl sulfate; TPCK, tosyl-L-phenyl chloromethyl ketone; TLCK, tosyl-L-lysyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; buffers A-I, see Materials and Methods; Mr, molecular weight. 3745The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.