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

Abstract

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

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

“…lOOK T antigen is larger than lytic SV40 T. Proteolytic breakdown of T antigens is likely to be the reason behind at least some earlier reports of multiple species of large T in lyticaily infected and transformed cells (1). Molecular weight values are of little use in comparing our results to previous studies of large T, since different gel systems yield different molecular weights for lytic T, and since none display large T at its calculated molecular weight of 81K (69).…”

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T‐antigen determined with a cleavage map

“…CV-1 cell culturees or primary mouse kidney cell cultures, grown to confluency in 85-mm plastic petri dishes in reinforced Eagle's medium containing 10 % fetal calf serum, were infected with SV40, and labeled with [35S]methionine (40-100 pCi/culture, 600-1200 Ci/mmol) as described [2]. SV40 mutant dl 1263 was kindly provided by Dr E. May (Villejuif, France).…”

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

“…In the one example presented herein, the SV40-specific RNA from virus-transformed human cells directs the synthesis of products, including the E-1 90,000 M, T-antigen polypeptide, similar to those directed by SV40 early mRNA extracted from lytically infected monkey cells. However, T-antigens isolated from different host species have been shown to vary in size (37,38). Moreover, variations in the virus cell-transformation system have been reported (39) as well as the isolation of revertant offspring of transformed cells, all of which can now be analyzed as to their virus-specific mRNA and related cell-free translation products.…”