The simian virus 40 large tumor antigen

In this review, I hope to achieve the following: (a) to document the presence of a lysosome-like proton pump ATPase in many different membrane systems of animal, plant and microbial origin; (b) to glean from the diverse data common characteristics of these ATPases, especially as regards their similarities and differences with mitochondrial-type F1F0 proton pump ATPases; and (c) to consider questions of synthesis and regulation of a cellular proton pump system with such a widespread distribution.     

Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen

Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum.     

Binding of simian virus 40 large tumor antigen to phospholipid vesicles

SV40 T antigen is the initiator protein of SV40 DNA replication. We examined the interaction of purified SV40 T antigen with phospholipids by (i) centrifugation analysis with phospholipid vesicles, (ii) filter binding assay and footprint analysis of T antigen binding to the replication origin of SV40 DNA and (iii) analysis of the initiation of SV40 DNA replication in vitro. In all cases, cardiolipin showed affinity for T antigen and inhibited its DNA binding capacity. Phosphatidylglycerol with unsaturated fatty acids also inhibited the binding of T antigen to the replication origin of SV40 DNA, whereas phosphatidylglycerol with saturated fatty acids did not. This finding suggested the importance of unsaturated fatty acids for the interaction of T antigen with phospholipids. Other phospholipids including phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine showed little or no affinity for T antigen.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

DNA-binding region of the simian virus 40 tumor antigen.

The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.     

Simian virus 40 large T antigen host range domain functions in virion assembly.

The simian virus 40 (SV40) T antigen host range mutants dl1066 and dl1140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants dl1066 and dl1140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with dl1066 and dl1140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40% sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants dl1066 and dl1140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests that the host range blockage may result from a failure of such mutants to add VP1 to 75S assembly intermediates.     

Relationship between simian virus 40 large tumor antigen expression and tumor formation in transgenic mice.

A line of transgenic mice containing the simian virus 40 (SV40) large tumor antigen gene under the control of the viral enhancer-promoter expressed this viral protein in the brains of these mice within the first 2 weeks after birth. Multiple foci of anaplastic cells formed in the choroid plexuses of these mice at 36 to 41 days after birth, and normal tissue coexisted with these transformed foci. Immunoperoxidase staining to detect the SV40 T antigen showed tumor-specific expression of nuclear T antigen at late times in tumor development, approximately 90 to 100 days and thereafter. The level of SV40 T antigen, on a per cell basis, appeared to be lower in the great majority of choroid plexus cells at earlier times in tumor development. These results suggest that low levels of tumor antigen (14 to 36 days) are present before detectable pathology (36 to 41 days) and the level of T antigen per cell is higher in rapidly growing late-stage tumors (older than 90 days).     

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.     

New properties of simian virus 40 large T antigen

An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40. The 8K T antigen was weakly labeled in vivo with [35S]methionine or 32Pi. A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen. The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen. Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin. The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences. Antibodies against large T antigen interacted with native recA protein.     

The carboxy terminus of simian virus 40 large T antigen is required to disrupt the yeast cell cycle

Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.     

Single strand DNA binding of simian virus 40 tumor antigen.

Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.     

The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions.

The large tumor antigen (T antigen) of simian virus 40 is necessary and sufficient for the neoplastic transformation of a number of established cell lines. Mutational analysis has revealed that a biochemical activity residing within the amino-terminal 121 amino acids of T antigen is sufficient to induce the transformation of some cell lines, such as C3H10T1/2. The same domain of the molecule also encodes the transactivation function of T antigen and the ability to complex with the retinoblastoma susceptibility gene product. However, the transformation of other lines, such as REF52, requires an additional activity that is affected by mutations in other portions of the molecule.     

Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the region coding for the carboxyl terminus of the T antigen.

Nucleotide sequence analysis of two simian virus 40 early mutants dl1263 and dl1265, which lack a DNA segment around map positions 0.21 and 0.18, respectively (C. Cole, T. Landers, S. Goff, S. MAnteuil-Brutlag, and P. Berg, J. Virol. 24:277--294, 1977), revealed in-phase deletions of 33 nucleotide pairs for dl1263 and 39 nucleotide paris for dl1265. The 33-base-pair deletion in dl1263 does not correspond to an apparent shortening by 6,000 daltons observed for the mutant T antigen. In dl1265 the normal termination signal as well as most of the proline-rich terminal tryptic peptide has been removed, and the carboxyl terminus of the mutant T antigen is a series of three cysteine residues.     

Purification of simian virus 40 large T antigen by immunoaffinity chromatography.

Simian virus 40 large T antigen from lytically infected cells has been purified to near homogeneity by immunochromatography of the cell extract on a protein A-Sepharose-monoclonal antibody column. The resulting T antigen retains biochemical activity; i.e., it hydrolyzes ATP and binds to simian virus 40 DNA at the origin of replication.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.     

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.