Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A

We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequencing of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PP2A). Identity was confirmed by comigration on two-dimensional (2D) gels and by 2D analysis of complete chymotryptic digests. In addition, PP2A-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 2D analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PP2A holoenzyme. Finally, the 36 kd PP2A catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PP2A and T antigens may be important for T antigen-mediated transformation.     

Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.     

Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. A two-dimensional gel analysis of SV40-, adenovirus-, and Kirsten murine sarcoma virus-transformed rat cells using the REF52 protein database

Two-dimensional gels of normal and virally transformed REF52 cells have been quantified and compared using the QUEST system for construction and analysis of protein databases. The REF52 protein map is based on more than 1600 high quality spots, and the relative amounts of these proteins are studied in 79 gels representing 12 major experiments. REF52 cells transformed by SV40, adenovirus, and Kirsten murine sarcoma virus (KiMSV) are compared to normal REF52 cells at several stages of growth from low density to confluence and after refeeding confluent cells. In addition, early (1-4 h) and late (21-24 h) responses to serum stimulation were measured in normal, SV40-and adenovirus-transformed cells. The database has been analyzed with respect to 1) known marker proteins and protein sets, 2) global comparison of protein patterns, and 3) selection of unknown spots which have interesting patterns of regulation. For the marker proteins, which include the tropomyosin family and the proliferation-sensitive nuclear antigen, new aspects of regulation by growth and transformation have been revealed. Proliferation-sensitive nuclear antigen, a protein known to be involved in DNA synthesis, is growth-regulated in normal cells and overexpressed in some SV40- and adenovirus-transformed cells. Global comparisons reveal no overall correlation between growth-regulated changes and transformation-induced changes; however, a set of 26 coregulated proteins, including proliferation-sensitive nuclear antigen, was found to be overexpressed in REF52 cells transformed by SV40 or adenovirus. These proteins are synthesized at rates that correlate with the rate of cell proliferation in REF52 and Kirsten murine sarcoma virus-transformed cells but, in SV40- and adenovirus-transformed cells, these proteins are synthesized at high levels independent of the rate of growth. These data suggest that the transforming proteins of SV40 and adenovirus share a function that results in deregulation of the genes coding for a class of cell cycle-regulated proteins.     

A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif

A 64 kd protein was shown to bind to RNAs that contain functional polyadenylation signals by a UV cross-linking procedure in which label was transferred from RNA substrate to protein in cell-free polyadenylation extracts. The 64 kd nuclear protein bound specifically to three different substrates (adenovirus type 5 L3, SV40 early, and SV40 late polyadenylation domains), as determined by competition experiments and partial protease analysis. Deleted derivatives of the SV40 late substrate that retained the sequence 5'-CUGCAAUAAACAAGUU-3' were able to bind the 64 kd polypeptide. This sequence contains the canonical AAUAAA element that has been shown to be indispensable for polyadenylation. A single nucleotide change, converting AAUAAA to AAGAAA, prevented binding of the 64 kd moiety. The 64 kd protein was shown to be distinct from poly(A) polymerase by biochemical fractionation.     

Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells.

The association of the cellular p53 protein with the E1B-55kd protein of adenovirus 5 (Ad5) is thought to result in inactivation of the p53 recessive oncogene product. Here we show that Ad5 E1-transformed 3Y1 rat cells which express low levels of the 55 kd E1B protein do not contain the p53-E1B 55kd complex. These cells have nuclearly located p53 and are highly oncogenic in nude mice. In 3Y1 cells expressing the E1B protein at a sufficiently high level, association between p53 and E1B-55kd occurs, resulting in an almost complete trapping of p53 into a discrete cytoplasmic body. These cells only form tumors after a very long latency period and in the tumors that eventually appear selection has occurred in favor of cells lacking the complex and containing free nuclear p53. Comparable results were found when highly oncogenic Ad12-transformed cells were supertransfected with the Ad5 E1B region. In none of the Ad-transformed mouse, rat and human cell lines examined, could we detect p53 of abnormal molecular weight or association with hsc70, neither could we immunoprecipitate p53 by the mutant specific antibody PAb240. These data suggest that a high level of nuclear p53 with a wild-type conformation contributes to the oncogenicity of Ad transformed cells.     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.     

Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells

Feldman, Lawrence A. (Baylor University College of Medicine, Houston, Tex.), Janet S. Butel, and Fred Rapp. Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells. J. Bacteriol. 91:813-818. 1966.-Adenovirus types 2, 7, and 12 undergo an abortive growth cycle in green monkey kidney cells; they induce the formation of adenovirus tumor antigen, but synthesis of adeno capsid antigen and infectious adenovirus was observed only when cultures were concomitantly infected with a simian papovavirus (SV40). Several other viruses, including herpes simplex and measles which replicate in monkey cells, and rabbit papilloma and human wart papovaviruses which do not, failed to stimulate adenovirus replication in the monkey cells. Adenovirus tumor antigen was detected 8 to 10 hr postinfection by immunofluorescent techniques. The antigen induced by adenovirus types 2 and 7 appeared as intranuclear masses; adenovirus type 12 tumor antigen also appeared as cytoplasmic and nuclear flecks. Sera from hamsters bearing tumors induced by adenovirus type 12 cross-reacted with tumor antigens induced by types 2 and 7 but not with antigens induced by SV40.     

Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation

Adenovirus 2 large plaque (Ip) mutants produce large clear plaques on human KB cells. These mutants are shown to be defective in inducing transformation of the established rat embryo cell line 3Y1. The Ip mutation was localized within one of the two transforming early gene blocks, E1b (map position 4.5 to 11.2) which codes for two major T antigens of 53 kd and 19 kd by marker transfer. The mutational defects in mutants Ip3 and Ip5 were analyzed by DNA sequence analysis and by analysis of viral E1 proteins. These results reveal that Ip3 and Ip5 mutations map within the 19 kd tumor antigen coding region. Mutant Ip3 has a single base pair change at the N terminus of 19 kd polypeptide, resulting in the substitution of valine for alanine. Mutant Ip5 has two mutational changes, one of which results in the substitution of tyrosine for aspartic acid near the N-terminal region. The second mutation changes the termination codon into a leucine codon, increasing the size of the 19 kd tumor antigen. These results provide direct genetic evidence for an essential role of the 19 kd tumor antigen in cell transformation and indicate that the N-terminal region of the 19 kd tumor antigen is an essential function domain for the induction of cell transformation.     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.