Protein kinase activity associated with simian virus 40 T antigen.

Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity.     

Interaction of partially purified simian virus 40 T antigen with circular viral DNA molecules.

Mixing chromatographic fractions containing simian virus 40 (SV40) T antigen with SV40 [3H]-DNA I (double-stranded, circular, supercoiled) results in the conversion of the nucleic acid to a form that will bind to a nitrocellulose filter. Unlabeled SV40 DNA I successfully competes with this reaction. Under the conditions employed, the antigen-containing fractions bind a variety of circular, viral DNA molecules. Chromatography of the antigen in three systems reveals that the T immunoreactivity migrates with DNA binding activity. In a kinetic heat inactivation experiment, the antigenic reactivity disappears simultaneously with the DNA binding activity. The data indicate the presence of a discernible DNA binding activity in fractions containing T antigen and suggest that the T antigen is the DNA binding protein being measured.     

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.     

Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.     

Enzymatic activities associated with a purified simian virus 40 T antigen-related protein

A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.     

T antigen binds to simian virus 40 DNA at the origin of replication.

A technique employing ferritin-conjugated antibody has been developed to visualize specific protein-DNA complexes in the electron microscope and has been used to demonstrate the preferential binding of simian virus 40 (SV40) T antigen at or near the origin of replication of SV40 DNA, 0.67 fractional length clockwise from the EcoRI restriction endonuclease cleavage site. urified covalently closed supercoiled circles of SV40DNA were treated with partially purified T antigen and the complex was stabilized by crosslinking with glutaraldehyde. Hamster antiT antigen gamma-globulin, ferritin-labeled goat anti-hamster gamma-globulin, and glutaraldehyde were then added sequentially. The location of the bound ferritin cores was measured with respect to the EcoRI cleavage site and the orientation of the cores relative to the ends of the DNA was determined with respect to the locations of Escherichia coli DNA unwinding protein, which binds to covalently closed supercoiled SV40 DNA at either of two preferred sites, 0.46 or 0.90 fractional length clockwide from the EcoRI cleavage site.     

Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.

Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei ofSV80 cells, a continuous line of simian virus 40-transformed human fibroblasts. A five-step purification method was used. Its apparent molecular weight (in sodium dodecyl sulfate/polyacrylamide gels) is approximately 90,000-94,000. It contains a detectable amino-terminal residue.     

Monomer molecular weight of T antigen from simian virus 40-infected and transformed cells.

T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.     

Purification of biologically active simian virus 40 small tumor antigen.

The simian virus 40 small tumor antigen (t antigen) gene has been cloned downstream from a hybrid Escherichia coli trp-lac promoter and a suitable ribosome binding site. A bacterial clone (865i) transformed by such a plasmid (pTR865) expresses this gene and, under optimal conditions, can produce greater than or equal to 5% of its total protein as t antigen. Soluble extracts of such a clone were relatively depleted in t antigen, which was found in the initial pellet fraction. The protein was recovered from this fraction in a significantly purified form by extraction with urea-containing buffer. After gel filtration of such t antigen-enriched solutions, highly purified protein was obtained. When either this fraction (freed of urea) or NaDodSO4 gel-purified 865i t antigen (rendered free of detergent) was injected into untransformed rat cells, dissolution of intracellular actin cable networks was observed.     

Differential affinities of simian virus 40 large tumor antigen for DNA.

The binding of simian virus 40 (SV40) large tumor antigen (T antigen) to DNA was analyzed by using the salt-sensitive affinities of the protein for various DNAs immobilized on cellulose. At least two types of interactions could be distinguished that differed in their stability. Higher salt concentrations were required to elute T antigen from SV40 DNA than from calf thymus DNA; and even greater salt concentrations were required for the lution of T antigen from multiorigin SV 40 DNA compared to wild-type SV40 DNA. This would indicate that T antigen can bind weakly or strongly to DNA, depending on the DNA sequence. It was also found that a greater proportion of rapidly labeled or newly synthesized T antigen binds more efficiently and tightly to multiorigin SV40 DNA than to long-labeled or older forms of T antigen. This approach can be utilized not only to distinguish between different forms of T antigens which vary in their affinities for DNA but also for rapidly obtaining highly enriched T antigen preparations.     

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT–CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT^Δ69–83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.Studies with simian virus 40 (SV40), a member of the Polyomaviridae family of tumor viruses, have led to fundamental insights into molecular processes of cell transformation and oncogenesis (1, 2). SV40 encodes the large tumor antigen (LT) with the potential to transform cells in culture and induce tumors in rodents. The tumorigenic features of SV40 have been attributed to binding and deactivation of key tumor suppressor proteins of the host cell including p53 and members of the retinoblastoma (pRB) family (1–3). In addition, SV40 LT was shown to be physically associated with Cullin 7 (CUL7; also named p185 or p193) (4, 5) as well as insulin receptor substrate 1 (IRS1) (6). It has been proposed that the association of SV40 LT with either CUL7 or IRS1 is critical to SV40 oncogenic transformation (7–9). However, the functional effect of LT interaction with CUL7/IRS1 and their pathophysiological interrelation remains mostly unknown.CUL7 is a scaffold protein responsible for assembling the multisubunit Cullin-RING E3 ubiquitin ligase 7 (CRL7) that consists of the RING-finger protein ROC1 and the Skp1-Fbw8 substrate-targeting subunit (10, 11). Genetic studies documented a pivotal growth-regulatory role of CRL7. Both cul7 (12) and fbw8 (13) null mice exhibit intrauterine growth retardation. In addition, CUL7 germ-line mutations were linked to 3-M syndrome, a hereditary disorder characterized by pre- and postnatal growth retardation in humans (14, 15), as well as Yakut dwarfism syndrome (16). DeCaprio and colleagues mapped the CUL7 interaction domain on SV40 LT to residues 69–83 and demonstrated that the CUL7 binding-deficient deletion mutant (LT^Δ69–83) lost its transformation potential despite maintaining its ability to bind and inactivate p53 and pRB members (8, 9). This suggested that CUL7 may act as a tumor suppressor and that constraining growth-inhibitory functions of CRL7 may be critical to SV40 transformation.We previously identified IRS1, a component of the insulin and insulin-like growth factor 1 (IGF1) signaling pathway, as a proteolytic target of CRL7 (17). Binding of insulin or IGF1 to its receptor induces tyrosine phosphorylation of IRS1 and subsequent activation of phosphatidylinositol-3-kinase (PI3K)/AKT and Erk mitogen-activated pathway kinase (MAPK) pathways (18). It was shown that CRL7-induced degradation of IRS1 is part of a negative feedback loop via mechanistic target of rapamycin complex 1 (mTORC1) to restrain IRS1 downstream signaling (17, 19). A more recent study suggested an mTORC2-dependent feedback inhibition of IRS1 by direct phosphorylation of Fbw8, resulting in enhanced stability of this F-box protein that promotes IRS1 degradation (20). Collectively, these studies have implicated roles for CRL7 in regulating both mTORC1 and mTORC2 signaling. Based on the above observations, we investigated whether SV40 LT impacts on CRL7 feedback regulation of IRS1 signaling in addition to its effects on p53 and pRB members.     

Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen.

Simian virus 40 large tumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.     

Thermolabile T (tumor) antigen from cells transformed by a temperature-sensitive mutant of simian virus 40.

Partially purified tumor (T) antigen from a strain of Chinese hamster lung cells transformed by wild-type simian virus 40 (SV 40) and either of two temperature-sensitive SV 40 mutants has been studied as a DNA binding protein. The DNA binding activity present in the T-antigen-containing fractions is inhibited by purified hamster anti-T IgG but not by equivalent amounts of nonimmune hamster IgG. T from either wild-type- or tsC219-transformed cells is relatively stable during heating at 44 degrees compared to T prepared from tsA239-transformed cells. These results strongly suggest that T is a product of the SV 40 A gene.     

In vitro formation of multimeric DNA structures mediated by purified simian virus 40 chromatin.

Simian virus 40 chromatin was incubated after purification by sucrose density-gradient centrifugation with various circular double-stranded DNA substrates. Monomeric rings were converted in the presence of Mg2+ to structures possessing a higher degree of complexity. Dimeric catenanes, as well as multimeric linear structures and concatemers, were generated, indicating that recombination events had occurred in vitro involving covalent linkage between different DNA molecules. Furthermore, apparently fused dimeric rings were observed. Their structures suggest that they may be recombination intermediates such as those described in a prokaryotic system [Potter, H. & Dressler, D. (1976) Proc. Natl. Acad. Sci. USA 73, 3000-3004]. Recombination did not take place between heterologous DNA substrates, as exclusively homologous multimeric DNA structures were observed.     

Specific in vitro adenylylation of the simian virus 40 large tumor antigen.

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).     

Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells.

Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.