Transformation of hamster kidney cells by BK papovavirus DNA.

Supercoiled BK papovavirus DNA was shown to transform hamster kidney cells using the calcium phosphate co-precipitation technique. The transformed cells contained intranuclear T-antigen(s) and rescuable virus and produced progressively growing tumors when inoculated into hamsters. A novel finding was the production in tumor-bearing animals of antinuclear antibody, which reacted against normal, untransformed cells; in addition, tumor serum contained antibody against virus-specific T-antigen(s).     

Loss of functional large T-antigen and free viral genomes from cells transformed in vitro by polyoma virus after passage in vivo as tumor cells.

We have analyzed the state, arrangement, and expression of polyoma viral DNA sequences in a number of in vitro-transformed Fischer rat cells before and after growth in vivo as tumour cells. When the in vitro lines used to induce the tumors contained only a single insert of viral sequences and did not produce either a full-size 100,000-dalton (100K) large T-antigen or free viral genomes, no differences in the above-mentioned properties were observed. By contrast, in vitro cell lines containing multiple inserts of viral sequences, a functional 100K large T-antigen, and free viral genome induced tumor cells which displayed a reduced number of inserts of viral sequences and which did not produce either a functional 100K large T-antigen or free viral genomes. All of the in vitro lines and their tumor cell derivatives expressed the polyoma virus 55K middle and 22K small T-antigen species. Possible mechanisms for the selection in vivo against cells containing a functional 100K large T-antigen and consequently free viral genomes are discussed.     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

Cell surface location of simian virus 40-specific proteins on HeLa cells infected with adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2.

HeLa cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 or Ad2+ND2 were analyzed for cell surface location of the SV40-specific hybrid virus proteins by indirect immunofluorescence microscopy. Two different batches of sera from SV40 tumor-bearing hamsters, serum from SV40 tumor-bearing mice, or two different antisera prepared against purified sodium dodecyl sulfate-denatured SV40 T-antigen, respectively, were used. All sera were shown to exhibit comparable T- and U-antibody titers and to specifically immunoprecipitate the SV40-specific proteins from cell extracts of Ad2+ND2-infected cells. Whereas analysis of living, hybrid virus-infected HeLa cells did not yield conclusive results, analysis of Formalin-fixed cells resulted in positive cell surface fluorescence with both Ad2+ND1- and Ad2+ND2-infected HeLa cells when antisera prepared against sodium dodecyl sulfate-denatured SV40 T-antigen were used as first antibody. In contrast, sera from SV40 tumor-bearing animals were not or only very weakly able to stain the surfaces of these cells. The fact that the tumor sera had comparable or even higher T- and U-antibody titers than the antisera against sodium dodecyl sulfate-denatured T-antigen but were not able to recognize SV40-specific proteins on the cell surface suggests that SV40 tumor-specific transplantation antigen may be an antigenic entity different from T- or U-antigen.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.     

Nuclear immunofluorescence by a monoclonal antibody against microtubule-associated protein-1 as it is associated with cell proliferation and transformation

Monoclonal antibody against microtubule-associated protein-1 produced intranuclear immunofluorescent spots, which disappeared under growth-inhibited conditions caused by serum starvation and saturated cell density in untransformed cells. A change of medium to 10% serum gave rise to the reappearance of nuclear spots before the resumption of DNA synthesis. This reversible change of immunofluorescence was also caused by a temperature shift in rat 3Y1 cells transformed by Simian virus-40-A640 (temperature-sensitive in large T-antigen). The fluorescence decreased during S phase of the cell cycle. In contrast the transformed cells always showed nuclear fluorescence, irrespective of serum concentrations or the cell cycle. Growth-inhibited cells previously treated with detergent and salt revealed nuclear fluorescent spots. This result suggested antigenic modification.     

Lymphotropic papovavirus transformation of hamster embryo cells

Hamster embryo cells were transformed by African green monkey lymphotropic papovavirus (LPV). The transformed cells contained intranuclear T-antigens demonstrable by fluorescent antibody staining with hamster anti-LPV serum. Analysis of uncloned and cloned lines of transformed cells for LPV sequences revealed that the viral DNA was present as free nonintegrated and integrated genomes; there were approximately 10 copies of free DNA and about one to two copies of integrated genomes per cell. The cells were highly tumorigenic when inoculated into hamsters and produced progressively growing tumors in 100% of newborn or 10-day-old hamsters that were inoculated with LPV-transformed cells. The serum from tumor-bearing hamsters reacted with LPV-transformed cells and also showed a weak reaction with simian virus 40-, BK virus-, and JC virus-transformed cells, thereby showing an antigenic relationship with the T-antigens of other primate polyomaviruses. The large T-antigen of LPV was found to be an 84,000-molecular-weight protein which was immunoprecipitated by hamster anti-LPV (antiviral) as well as by tumor serum.     

Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.     

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.     

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture.     

Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction

A heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of the monoclonal antibodies F4, F5, and LT1 directed against mouse polyomavirus large T-antigen. Phage selected by biopanning was cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay. In phage reacting with the F5 antibody the deduced amino acid sequence of the displayed peptides corresponded to a segment of large T-antigen. In phage reacting with the antibodies F4 and LT1, no such similarity was observed. The kinetics of phage particle-monoclonal antibody complex formation and dissociation was analyzed in an optical biosensor instrument. Sensor chips of standard quality were useful for binding analysis of T7 phage in crude lysates of infected Escherichia coli. We synthesized peptides corresponding to selected consensus sequences and showed by biosensor analysis that these peptides (linear NH3-CPNSLTPADPTMDY-COOH and NH3-NSLTPCNNKPSNRC-COOH with an intramolecular S--S bridge) were able to compete with large T-antigen in binding to the corresponding antibodies (LT1 and F4). These synthetic peptides were also used for gentle and specific dissociation of large T-antigen-antibody complexes. The results demonstrate the potential of phage T7 for display of peptides and for rapid analysis of interactions of these peptides with ligands.     

The chicken ovalbumin promoter is under negative control which is relieved by steroid hormones.

Steroid hormone regulation of activity of the chicken ovalbumin promoter was studied by microinjection of chimeric genes into the nuclei of primary cultured oviduct tubular gland cells. The chimeric genes contained increasing lengths of ovalbumin gene 5'-flanking sequences fused to the sequence coding for the SV40 T-antigen. Promoter activity was estimated by monitoring synthesis of T-antigen. The activity of the ovalbumin promoter is cell-specifically repressed in these oviduct cells and the repression is relieved upon addition of steroid hormones. The -132 to -425 region of the ovalbumin promoter which is responsible for this negative regulation behaves as an independent functional unit containing the regulatory elements necessary for both repression (in the presence of steroid hormone antagonists) and induced derepression (in the presence of steroid hormones) of linked heterologous promoters.     

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

Synthetic peptides containing a region of SV40 large T-antigen involved in nuclear localization direct the transport of proteins into the nucleus

We studied the mechanism of transport of proteins into the nucleus using synthetic peptides containing the nuclear location signal sequence of Simian virus 40 (SV 40) large T-antigen. When chick erythrocytes containing a synthetic large T-antigen nuclear translocation signal were fused with SV 40-transformed human fibroblasts, the migration of native large T-antigen into the chick nuclei was suppressed. Migration of proteins detected by human specific antinuclear autoimmune antibody was not blocked. An analog of the nuclear location signal peptide did not inhibit entry of large T-antigen into the chick nuclei. This result suggests that the peptide blocked the migration of only native large T-antigen into the nucleus, and that the signal of the majority of nuclear proteins for nuclear transport is not the same as that of the large T-antigen. The synthetic peptide was conjugated chemically with bovine serum albumin (BSA) and introduced into the cytoplasm of cultured human cells by red blood cell ghost-mediated microinjection. The BSA-synthetic peptide conjugate was found predominantly in the nucleus within 2 h after its introduction into the cells. BSA conjugated with the cross-linking reagent alone was not transported into the nucleus. Acetylated synthetic peptide was not effective in promoting nuclear localization of BSA. Mild trypsin treatment of the BSA-synthetic peptide conjugate suppressed nuclear localization. Conjugates of the synthetic peptide with phycoerythrin (Mr about 150 kD) and with secretory IgA (Mr about 380 kD) were both found in the nucleus very shortly after their introduction into the cytoplasm. These results suggest that the synthetic peptide containing the nuclear location signal sequence provides exogenous proteins with the ability to migrate into the nucleus. But, since a conjugate of the synthetic peptide with IgM (Mr about 940 kD) did not migrate into the nucleus after its microinjection, there may be a size limit in nuclear transport of conjugated proteins.     

Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding

We have constructed a series of deletion mutants in the lysozyme promoter region fused to the SV40 T-antigen coding region. Regulated expression was tested after microinjection of the lysozyme deletion mutants into primary cultures of chicken oviduct cells using fluorescent antibodies against T antigen. Deletion of lysozyme gene sequences upstream of position - 164 was accompanied by loss of both progesterone- and glucocorticoid-induced expression. Using the rat liver glucocorticoid receptor for binding studies, two separate binding sites have been identified: a strong binding site that is destroyed by deletion of lysozyme sequences between positions -74 and -39 and a weaker binding site contained between positions -208 and -161 upstream of the lysozyme cap site.