Phosphorylation pattern of large T antigens in mouse cells infected by simian virus 40 wild type or deletion mutants

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been extensively studied in productive infection of monkey cells. In this study, we analyzed the phosphorylation sites of large T antigen from SV40-infected nonpermissive mouse cells by partial proteolysis fingerprints and analysis of the phosphoamino acids present in the resulting fragments. The wild-type virus and deletion mutants (dl1263, dl1265, dl2194, and dl2198) were used for infection. On the basis of our results and published data (M. Schwyzer, R. Weil, and H. Zuber, J. Biol. Chem. 225:5627-5634, 1980), a cleavage map of large T antigen was established. It was reported that at least four sites of phosphorylation were present. The amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the prolinerich C-terminal end of the molecule at position 701 or 708. On the basis of the concensus as to the amino acid sequence surrounding the recognition sites for protein kinases, it was possible to more precisely locate this phosphothreonine at residue 701. Moreover, the C-terminal part of the molecule contained phosphoserine at a more internal position. In addition, this study firmly established the presence of a phosphothreonine in the N-terminal part of large T antigen. In conclusion, it was shown that the location of phosphorylation sites of large T antigen produced by nonpermissive mouse cells infected by SV40 is strikingly similar to that reported by other groups for large T antigen produced by SV40-infected permissive cells.     

Characterization of different tumor antigens present in cells transformed by simian virus 40.

In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.     

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.     

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.     

Cytoplasmic T antigens of mouse and human cells transformed by a simian virus 40 tsA mutant

Simian virus 40 T antigens accumulate in the cytoplasm of simian virus 40 tsA207 transformants of primary mouse kidney or human retinoblastoma cells grown at 40 degrees C in 10% serum.     

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.     

Association of phosphorylated simian virus 40 T-antigen with subnuclear fractions of infected and transformed cells

To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.     

Structure of integrated simian virus 40 DNA in transformed mouse cells

The structure of integrated viral DNA sequences in four lines of simian virus 40 (SV40)-transformed Balb/c 3T3 cells has been probed using restriction endonucleases and the Southern (1975) transfer method. By considering data from a large number of restriction digests of DNA from each line, and by using a novel method of handling the data, we have constructed fairly detailed physical maps of the integrated DNA in each line. The most striking of the features of the maps described here is that none is easily explained by the integration of a single SV40 genome into the DNA of the host cell. Three of the lines contain at least two distinct integrated segments and the fourth contains a single segment longer than the viral DNA. Considered individually, only two of the seven segments that we have mapped might be unit length. Of the remaining five, two are longer and three are shorter than the viral genome. It seems likely, therefore, that at least in SV40-transformed Balb/c 3T3 cells simple, single integrations are rare.The endpoints of these seven segments of integrated DNA fall at many positions distributed over the entire genome, confirming earlier studies (Ketner &; Kelly, 1976; Botchan et al., 1976), which indicated that SV40 integration is not absolutely site-specific.Finally, one of the lines mapped here (SVB209) does not possess an intact SV40 early region, an observation that suggests the possibility that a normal viral large T polypeptide is not synthesized by this line.     

The arrangement of simian virus 40 sequences in the DNA of transformed cells.

High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome.     

DNA polymerase activities in growing cells infected with simian virus 40.

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.     

Human thymic epithelial cells are frequently transformed by retroviral vectors encoding simian virus 40.

The thymic microenvironment contains a mixture of phenotypically distinct epithelial cells of varied functions, some of which are unknown. In an attempt to understand their relevance to T cell differentiation in the thymus, human thymic epithelial cell clones from both fetal (SM3-SM5) and postnatal (SM6) thymus were produced by using a defective recombinant retroviral vector encoding the simian virus 40 large T antigen and the neomycin resistance gene. The presence of keratins 8 and 18, desmosomes, and tonofilaments confirmed the epithelial origin of the cell strains. The cells expressed Thy-1 and HLA-Class I at high levels, showed weak-expression antigens defined by TE3B and A2B5, and low to negligible levels of the MR19-defined molecule. When compared with the phenotype of thymic epithelial cells in situ, the cell strains appear to be derived from neuroendocrine components in the outer cortical region of the human thymus. The use of retroviral vectors to transform human thymic epithelium was considerably more efficient than transfection with a plasmid carrying the origin of replication-defective SV40 large T gene. In the latter case, only two cell strains with subcapsular epithelial phenotypes were derived from fetal thymus. With the retroviral vectors, epithelial cell strains could, for the first time, be generated from human postnatal thymus as well as from fetal thymus.     

Altered metabolism of heparan sulfate in simian virus 40 transformed cloned mouse cells.

Glycoconjugates have been analyzed from a family of closely related mouse cells: a parent clone and three daughter subclones, two of which expressed the simian virus 40 (SV40) T-antigen. The experimental procedure involved the simultaneous comparison by DEAE-cellulose chromatography of papain-digested macromolecules from the parent, labeled with [3H]glucosamine, and one of the daughter subclones, labeled with [14C]-glucosamine. Three cultures compartments (the medium, the cell surface trypsinate, and the cells) from the paired cell lines were combined at the earliest time during the harvesting of the cells. Heparan sulfate on the surface of cells and secreted into the medium from T-antigen-positive subclones was eluted at lower salt concentrations from the anion exchange column than that from the parent clone. In the viable trypsinized cells a marked reduction of heparan sulfate was detected in the T-antigen-positive subclones. These changes were highly reproducible, were observed during both logarithmic and stationary phase of growth, and neither change was observed in the T-antigen-negative sister subclone. The elution point of heparan sulfate from Sepharose 6B was unaltered. Ratios of 35S to 3H for heparan sulfate obtained from cells doubly labeled with [35S]sulfate and [3H]glucosamine were lower in the T-antigen-positive subclones. Similar changes for the 35S to 3H ratio of chondroitin sulfate were associated with only small alterations in elution from anion exchange columns. Kinetic experiments suggested a reduced rate of incorporation of [35S]sulfate with no change in turnover rate. A substantial portion of the labeled heparan sulfate was associated with the cell surface; in contrast most of the hyaluronic acid and a large proportion of the chondroitin sulfate was apparently secreted. Quantitative changes in hyaluronic acid labeling did not correlate with expression of T-antigen. Glycosaminoglycans left on the dish after detaching cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid were nearly completely released by subsequent trypsinization. Cell detachment by trypsinization left an insignificant amount of labeled glycosaminoglycan on the dish surface. The alterations in heparan sulfate metabolism correlated with the expression of T-antigen and with the cells' ability to grow to high densities in monolayer culture, but not with growth in suspension in viscous medium. Tumorigenicity of the subclones was essentially the same as that of the parent clone.     

Collagenase production by immortalized human aortic endothelial cells infected with simian virus 40.

Human aortic endothelial cells, isolated at autopsy from a 52-year-old male dying from lung cancer, were treated with simian virus 40 (SV40). One colony was isolated from the infected endothelial cell culture 4 weeks after infection. The cells expressed SV40 large T antigen and p53 protein (p53) in their nuclei but lacted the characteristics of a transformed phenotype. The cells grew well in a monolayer over the 97th passage and exhibited Factor VIII-related antigen, Ulex europaeus 1 agglutinin (UEA-1) as endothelial cell markers, and a well-developed fibronectin network. The amount of prostacyclin synthesized by the cells was less than the amount synthesized by normal aortic or umbilical cord vein endothelial cells. The cells produced relatively large amounts of procollagenase, and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) augmented the ability of the cells to produce this enzyme. These immortalized human aortic endothelial cells, which have some characteristics of normal endothelial cells and, like capillary endothelial cells, have the ability to produce collagenase, will probably prove useful for studies of atherosclerosis and angiogenesis.     

Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used.