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.     

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.     

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).     

Nonselective expression of simian virus 40 large tumor antigen fragments in mouse cells.

To understand the role of various functional domains of simian virus 40 early tumor antigens, we have cloned and introduced into mouse cells portions of early simian virus 40 DNA. Two types of truncated large tumor antigen (33 and 12.3 kilodaltons), as well as small tumor antigen, were identified by immunoprecipitation. Both truncated large tumor antigens have been found to be overproduced with respect to the small tumor antigen, although the 12.3-kilodalton truncated large tumor antigen was more stable than the 33-kilodalton one. Nonviral 53-kilodalton protein was not found associated with either truncated large tumor antigen in immunoprecipitations.     

Nature and origin of the RNA associated with simian virus 40 large tumor antigen.

Simian virus 40 (SV40) large tumor (T) antigen isolated from mammalian cells undergoing lytic or transforming infection is associated with small RNA fragments ("T-antigen RNA") that are protected from nuclease digestion. The rather high complexity of the ribonuclease T1 fingerprints of T-antigen RNA suggested that it is mainly derived from cellular heterogeneous nuclear RNAs. In the present study, 5'-32P-labeled T-antigen RNA was hybridized to monkey, mouse, and human Alu and SV40 DNA, and the nucleotide sequence of 37 T1 oligonucleotides was determined. The results suggest that the bulk of T-antigen RNA is derived from noncoding, double-stranded, ordered regions of cellular heterogeneous nuclear RNAs that exhibit sequence homologies with interspersed repetitive elements of the cellular genome. The possible biological implications of these results are discussed.     

Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen.

Antibodies specific for the amino- and carboxy-terminal portions of simian virus 40 large tumor (T) antigen were obtained by immunization of rabbits with synthetic peptides corresponding to these regions. The amino-terminal synthetic peptide has the sequence Ac-Met-Asp-Lys-Val-Leu-Asn-Arg-(Tyr). The tyrosine residue was introduced in order to couple the peptide to bovine serum albumin with bis-diazotized benzidine. The carboxy-terminal peptide has the sequence Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr. It was coupled to bovine serum albumin with glutaraldehyde. The antisera against both peptides reacted with large T antigen. The specificity of the immune reaction was demonstrated by inhibition experiments using excess synthetic peptides. Furthermore, fragments of T antigen encoded by the nondefective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4, which contain the carboxy terminus and lack the amino terminus of large T antigen, were precipitated only with antiserum to the carboxy-terminal peptide. Small T antigen was not precipitated with either serum, suggesting that the amino terminus of small T antigen has a conformation different from that of large T antigen or that it is sterically hindered by a host protein. The procedures used here are of general importance for identification and characterization of gene product.     

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.     

Signal-mediated nuclear transport in simian virus 40-transformed cells is regulated by large tumor antigen.

Transformation of cultured cells with simian virus 40 (SV40), or transfection with the early region of the SV40 genome, causes a significant increase in both the rate of signal-mediated nuclear transport and the functional size of the transport channels (located in the pore complexes). By microinjecting purified large tumor (T) antigen into the cytoplasm of murine BALB/c 3T3 cells, we have demonstrated that this protein alone can account for the increase in transport capacity. The T antigen-dependent changes can be partially inhibited by cycloheximide and require a functional nuclear localization sequence. Although necessary, the nuclear localization sequence by itself cannot produce the observed variations in nuclear permeability and presumably function in a "helper" capacity, in association with another, as yet unidentified domain.     

The tsA58 simian virus 40 large tumor antigen disrupts megakaryocyte differentiation in transgenic mice.

Thrombocytopenia is a condition of multiple etiologies affecting the megakaryocyte lineage. To perturb this lineage in transgenic mice, the tsA58 mutation of the simian virus 40 large tumor antigen was targeted to megakaryocytes using the platelet factor 4 promoter. Ten of 17 transgenic lines generated exhibited low platelet levels, each line displaying a distinct, heritable level of thrombocytopenia. Within a line, the degree of the platelet reduction correlated directly with transgene zygosity. The platelet level could be further reduced by the inactivation of one copy of the endogenous retinoblastoma gene. Western blot analysis detected large tumor antigen protein in the most severely affected lines; less affected lines were below the level of detection. Platelets and megakaryocytes from thrombocytopenic mice exhibited morphological abnormalities. Mice with either normal or reduced platelet levels developed megakaryocytic malignancies with a mean age of onset of about 8 months. There was no correlation between severity of thrombocytopenia and onset of malignancy. These mice provide a defined genetic model for thrombocytopenia, and for megakaryocytic neoplasia, and implicate the retinoblastoma protein in the process of megakaryocyte differentiation.     

Template supercoiling during ATP-dependent DNA helix tracking: studies with simian virus 40 large tumor antigen.

Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase 1 in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins.     

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.     

Biological activity of purified simian virus 40 T antigen proteins.

Proteins related to simian virus 40 (SV40) T antigen uere isolated from cells infected with adenovirus 2/SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000- and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.     

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.     

ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication.

Simian virus 40 (SV40) large tumor antigen (T antigen) binds to two contiguous sites at the SV40 origin of replication. Of these two sites, I and II, only site II is critical for replication. We have studied the interaction between T antigen and these sites by two methods--nitrocellulose filter binding and DNase I protection. We show that T antigen binds with high occupancy to site I at 0 degrees C, 25 degrees C, and 37 degrees C but to site II only at 0 degrees C and 25 degrees C. At 37 degrees C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T antigen and site II. ATP can be replaced efficiently by adenosine 5'-[beta,gamma-imido]triphosphate and ADP, suggesting that hydrolysis of the nucleotide is not essential for the binding of T antigen to site II. The binding to the region critical for replication can occur in the presence of a variety of nucleoside triphosphates; dATP supports binding at a concentration 1/30th that of ATP, while dGTP and rGTP were inactive at all concentrations tested.     

Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.     

A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus.

We have constructed a frameshift mutation in the simian virus 40 early region using a novel method of oligonucleotide-directed mutagenesis. The mutated DNA specifies an 84,000-dalton large tumor antigen that consists of approximately equal to 75,000 daltons encoded by the wild-type reading frame and 9,000 daltons, by the alternative reading frame (wild-type large tumor antigen is approximately equal to 82,000 daltons). The frameshifted carboxyl terminus of the protein bears a strong similarity to the same region of polyoma virus middle-sized tumor antigen. We have found that the mutant DNA is unable to replicate when introduced into permissive monkey cells and incapable of transforming nonpermissive mouse cells.