Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Presence in growth-arrested cells of cellular proteins that interact with simian virus 40 small-t antigen.

Two cellular proteins, 56K and 32K, found in association with simian virus 40 small-t antigen were not induced by viral infection. In addition, the proteins were expressed by cells in the growth arrest period, a time in which small-t function is of importance in infection and transformation.     

Simian virus 40 small-t antigen binds two zinc ions.

Six cysteine residues of the simian virus 40 small-t antigen (small-t) are important for stability of the protein. Stability has been shown to be related to the ability of small-t to bind zinc ions in vitro. Purified small-t expressed either in bacteria or from baculovirus vectors binds two molecules of zinc per molecule of protein. Thus, small-t may resemble GAL4, which contains a Zn(II)2Cys6 binuclear cluster.     

Effect of zinc ions on the biochemical behavior of simian virus 40 small-t antigen expressed in bacteria.

The simian virus 40 small-t antigen contains 10 cysteine residues, 6 of which are organized in two CysXCysXXCys clusters. Mutation of individual Cys residues in the two clusters or mutation of specific residues found between these clusters causes pronounced instability of the protein in animal cells. Protein instability correlates with failure of the bacterially expressed mutant proteins to bind zinc ions, an interaction which allows purification of large amounts of small-t antigen in monomeric form.     

Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex.

Two cellular proteins, 61 and 37 kDa, are found in association with the simian virus 40 (SV40) small-t antigen. Fractionation in standard chromatography systems showed that these proteins were associated with one another in uninfected cells, suggesting that the small-t antigen may bind the complex as a whole and not each individual protein independently. In the presence of N-ethylmaleimide, the 37-kDA protein was selectively released from immune complexes, leaving the small-t antigen and 61-kDa protein in association. This result suggests that the small-t antigen may bind only the 61-kDa protein and that the 37-kDa protein may be associated with immune complexes by virtue of its association with the 61-kDa cellular protein.     

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.     

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.     

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.     

Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells.

Two cellular proteins are found to be complexed with simian virus 40 small-t antigen in cellular extracts. The complex is a relatively unstable but dynamic one which can dissociate and reform in extracts. In extracts of permissive monkey kidney cells, the small-t antigen appeared to be present in excess, whereas the cellular proteins were nearly entirely committed to the complex in permissive monkey kidney cells.     

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.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.     

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.     

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.     

Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function

Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.     

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.     

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.     

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.     

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.