Density dependent inhibition of both growth and T-antigen expression in revertants isolated from simian virus 40-transformed mouse SVT2 cells.

Phenotypic revertants were isolated from simian virus 40-transformed cells in order to examine the relationship between simian virus 40 T-antigen expression and G1 arrest of growth. Revertant clones with increased adherence were selected from cultures of SVT2, a simian virus 40-transformed BALB/c mouse cell line, and screened to find arrestable revertant clones which inhibited DNA synthesis when crowded. The clones selected from untreated SVT2 were unstable and showed little or no inhibition of DNA synthesis when crowded. Stable revertants were found after treatment of SVT2 with Colcemid to increase ploidy. The stable revertants all lost most transformed growth properties tested, including tumorigenicity, but only a few showed the same degree of inhibition of DNA synthesis at high cell density as BALB/3T3. All revertant clones expressed T antigen at low cell density. Three revertants showed coordinate inhibition of DNA synthesis and apparent loss of T antigen at high cell density. We suggest that changes in gene dosage rather than mutations caused the altered properties of the new revertants and that continued DNA synthesis in confluent cultures may be the transformed phenotype that requires the least simian virus 40 T antigen.     

Measurements of the molecular size of the simian virus 40 large T antigen.

A measure of the molecular weight of the large simian virus 40 T antigen was sought by SDS-polyacrylamide gel electrophoresis, random-coil chromatography, and sedimentation-velocity analysis in a density gradient. Large T antigen obtained from a simian virus 40-transformed human cell line either by immunoprecipitation or by standard preparatory methods migrated like a 94,000-molecular-weight (approximately 94K) polypeptide in SDS-gels but was found to have an approximate was observed with T antigen obtained from lytically infected monkey cells. In view of the strong theoretical basis for the guanidine method and the agreement with the sedimentation data, these findings suggest that the molecular weight of this protein is approximately 75 to 80K as opposed to 94 to 100K and, therefore, that considerably less than the entire early region of simian virus 40 is required to encode it. This size estimate is in keeping with earlier results which revealed a normal-size T antigen in cells infected with viable deletion mutants lacking as much as 10% of the early region.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

Use of simian virus 40 large T-beta-galactosidase fusion proteins in an immunochemical analysis of simian virus 40 large T antigen.

Simian virus 40 large T antigen is a multifunctional protein that is encoded by the early region of the viral genome. We constructed fusion proteins between simian virus 40 large T antigen and beta-galactosidase by cloning HindIII fragments A and D of the virus into the HindIII sites of expression vectors pUR290, pUR291, and pUR292. Large amounts of the fusion protein were synthesized when the DNA fragment encoding part of simian virus 40 large T antigen was in frame with the lacZ gene of the expression vector. Using Western blotting and a competition radioimmunoassay, we assessed the binding of existing anti-T monoclonal and polyclonal antibodies to the two fusion proteins. Several monoclonal antibodies reacted with the protein encoded by the fragment A construction, but none reacted with the protein encoded by the fragment D construction. However, mice immunized with pure beta-galactosidase-HindIII fragment D fusion protein produced good levels of anti-T antibodies, which immunoprecipitated simian virus 40 large T antigen from lytically infected cells, enabling derivation of monoclonal antibodies to this region of large T antigen. Therefore, the fusion proteins allowed novel epitopes to be discovered on large T antigen and permitted the precise localization of epitopes recognized by existing antibodies. The same approach can also be used to produce antibodies against defined regions of any gene.     

Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.     

Differential phosphorylation of cytoplasmic and nuclear variants of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses.

The phosphorylation patterns of cytoplasmic and nuclear forms of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses were analyzed by two-dimensional peptide mapping. The PARA(cT) mutant which encodes a large T antigen defective for nuclear transport was used as source for cytoplasmic large T antigen. The data suggest that the large T antigen is phosphorylated in a sequential manner at a subset of sites in the cytoplasm and at additional sites in the nucleus.     

Complex formation of simian virus 40 large T antigen with cellular protein p53

We investigated the formation of native complexes between simian virus 40 large T antigen and the cellular protein p53 (T-p53) by using simian virus 40 tsA58-transformed mouse fibroblasts (tsA58 F2b). We observed that newly synthesized p53 bound to all structural subclasses of large T antigen detectable on sucrose density gradients. This led to various intermediates of T-p53 complexes which converted within 2 h into typical mature aggregates. The final levels of stable T-p53 complexes seemed to be determined by p53 rather than by large T antigen.     

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.     

Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Simian virus 40 large T antigen stably complexes with a 185-kilodalton host protein.

Stable interactions between simian virus 40 large T antigen and host proteins are believed to play a major role in the ability of the viral protein to transform cells in culture and induce tumors in vivo. Two of these host proteins, the retinoblastoma susceptibility protein (pRB) and p53, are products of tumor suppressor genes, suggesting that T antigen exerts at least a portion of its transforming activity by complexing with and inactivating the function of these proteins. While analyzing T antigen-host protein complexes in mouse cells, we noted a protein of 185 kDa (p185) which specifically coimmunoprecipitates with T antigen. Coimmunoprecipitation results from the formation of stable complexes between T antigen and p185. Complex formation is independent of the interactions of T antigen with pRB, p120, and p53. Furthermore, analysis of T-antigen mutants suggests that T antigen-p185 complex formation may be important in transformation by simian virus 40.     

Malawi Polyomavirus Is a Prevalent Human Virus That Interacts with Known Tumor Suppressors

Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.     

Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.     

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.     

Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.     

Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.