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.     

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.     

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 zinc finger region of simian virus 40 large T antigen

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence Cys-302LeuLysCys-305IleLysLysGluGlnProSerHisTyrLysTyrHis- 317GluLysHis-320. In a conventional representation, the binding of zinc to the cysteines and histidines at positions 302, 305, 317, and 320 would form two minor loops and one major loop from the intervening amino acids. We made single amino acid substitutions at every position in the finger to identify possible functional elements within the putative metal-binding domain. Amino acids in the zinc finger could be divided into three classes characterized by distinct roles in DNA replication and transformation. Class 1 consisted of amino acids in the two minor loops of the finger and in the amino-terminal part of the major loop. Mutations here did not affect either replication or transformation. Class 2 consisted of the SerHisTyrLysTyr amino acids located in the carboxy terminus of the major loop of the finger. Mutations in this contiguous region reduced replication of the mutant viruses to different degrees. This clustering suggested that the region is an active site important for a specific function in DNA replication. With the exception of a mutation in the histidine at position 313, these mutations had no effect on transformation. Class 3 consisted of the proposed zinc-binding amino acids at positions 302, 305, 317, and 320 and the histidine at position 313 in the major loop of the finger. Mutations in these amino acids abolished the viability of the virus completely and had a distinctive effect on the transforming functions of the protein. Thus, the five cysteines and histidines of class 3 may play an important role in determining the overall structure of the protein. The histidine at position 313 may function both in the active site where it is located and in cooperation with the proposed zinc-binding ligands.     

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.     

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.     

Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen

Simian virus 40 (SV40) large tumor antigen (T antigen) exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts from infected monkey cells. Three subclasses of this antigen from SV40-infected monkey cells have been separated and characterized: the 5S, 7S, and 14S forms. Newly synthesized T antigen occurs primarily in the 5S form. Chemical cross-linking provided evidence that the 14S form is primarily a tetramer, whereas the 5S and 7S forms could not be cross-linked into oligomers. The DNA-binding properties of each subclass were investigated after immunopurification. The affinities of the three forms for SV40 DNA and for a synthetic 19-base-pair sequence from binding site I are very similar (equilibrium dissociation constant [KD], 0.3 to 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S subclasses increased sharply at about 40 h after infection, whereas the activity of the 14S subclass was maintained at a constant low level throughout infection. A model relating oligomerization and DNA binding of T antigen in infected cells is presented.     

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.     

Mapping the transcriptional transactivation function of simian virus 40 large T antigen

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.     

The finger domain of simian virus 40 large T antigen controls DNA-binding specificity.

The specificity and regulation of protein-DNA interactions play a crucial role in all aspects of communication between genotype and phenotype in a cell. The large T antigen of simian virus 40 binds to identical, yet quite differently arranged, pentanucleotide motifs in the simian virus 40 control region, sites I and II. Wild-type T antigen preferentially binds site I. We demonstrate that a bacterial peptide encoding residues 1 to 259 (T260) includes the essential amino acids required for binding to both DNA sites but predominantly binds site II. However, a longer peptide (residues 1 to 369; T370) binds almost exclusively to site I. Thus, the addition of amino acids 260 to 369 to the T260 peptide results in the loss of site II binding. This region includes a single putative metal-binding region, and mutation of T370 at either conserved cysteine of the finger results in equal but inefficient binding to both sites. While no metal binding has been shown to be directly associated with this sequence, these results suggest a novel, perhaps structural, function for such a finger motif, since this domain of T antigen appears to play a crucial role in modulating the DNA-binding behavior of T-antigen peptides.     

Improved localization of phosphorylation sites in simian virus 40 large T antigen.

The location of phosphorylation sites in the large T antigen of simian virus 40 has been studied both by partial chemical cleavage and by partial proteolysis of various forms of large T. These included the full-size wild-type molecule with an apparent molecular weight of 88,000, deleted molecules coded for by the mutants dl1265 and dl1263, and several shortened derivatives generated by the action of a cellular protease. These molecules differed from each other by variations in the carboxy-terminal end. In contrast, a ubiquitous but minor large T form with a molecular weight of 91,000 was found to be modified in the amino-terminal half of the molecule. In addition to the phosphorylation of threonine at position 701 (K.-H. Scheidtmann et al., J. Virol. 38:59-69, 1981), two other discrete domains of phosphorylation were recognized, one at either side of the molecule. The amino-terminal region was located between positions 81 and 124 and contained both phosphothreonine and phosphoserine residues. The carboxy-terminal region was located between approximate positions 500 and 640 and contained at least one phosphoserine residue but no phosphothreonine. The presence in the phosphorylated domains of large T of known recognition sequences for different types of protein kinases is discussed, together with possible functions of large T associated with these domains.     

Metabolic turnover of phosphorylation sites in simian virus 40 large T antigen.

Four (groups of) phosphorylation sites exist in the large T antigen of simian virus 40, and they involve at least two serine and two threonine residues (Van Roy et al. J. Virol. 45:315-331, 1983). All the phosphorylation sites were found to be modified and again dephosphorylated at discrete rates, with phosphoserine residues having the highest turnover rate. The measured half-lives ranged between 3 h (for the carboxy-terminal phosphoserine site) and 5.5 h (for the amino-terminal phosphothreonine site). The influence of four temperature-sensitive A mutations on phosphorylation of large T antigen was also examined. At restrictive temperature, phosphorylation of the carboxy-terminal phosphoserine in mutated large T antigen was found to be particularly impaired. These data emphasize the physiological importance of the latter phosphorylation site.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.     

Evidence for transmembrane orientation of acylated simian virus 40 large T antigen.

In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.     

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.     

Self-assembly of simian virus 40 large T antigen oligomers by divalent cations.

In simian virus 40-transformed cells, simian virus 40 large T antigen can be detected in different forms separable by sucrose density gradient centrifugation. In our experiments, light forms sedimented around 5 to 7S, oligomers such as tetramers were detected around 16S, and higher aggregates sedimented in a broad distribution reaching above 23S. The oligomers sedimenting at and above 16S could be disassembled into the slowly sedimenting 5 to 7S forms by chelating agents [EDTA or ethylene bis(oxonitrilo)tetraacetate]. After the addition of divalent cations (CaCl2 or MgCl2) in excess of chelating agents, oligomeric forms reassembled and appeared in a sedimentation pattern resembling that observed before treatment with chelating agents. Time course studies permitted the identification of the 5 to 7S forms as precursors upon pulse-labeling (15 min); the 16S and higher oligomers were identified as the successors after a 14-h chase. Treatment of extracts of pulse-chase-labeled cells with chelating agents again disassembled the oligomers, whereas pulse-labeled precursors did not change their 5 to 7S sedimentation pattern. Adding an excess of divalent cations reassembled the pulse-chase-labeled T antigen to oligomers but did not influence the sedimentation behavior of pulse-labeled 5 to 7S precursors. It is therefore reasonable to assume that a posttranslational modulation induces divalent cation binding, leading finally to the oligomerization of T antigen. Thus, some of the multifunctional activities of T antigen can be dictated by divalent cation binding properties.     

Stoichiometry of large T antigen and pp53 in complexes isolated from simian virus 40-transformed rat cells.

Simian virus 40-transformed cells synthesize high-molecular-weight protein complexes (22 to 30S) that consist of the virus-coded large T antigen (81,500 daltons) and the cellular antigen pp53. These complexes were partially purified from lysates of transformed rat cells by sucrose velocity sedimentation. The stoichiometry of the two proteins in the complex was studied by direct enzyme-linked immunosorbent assays, using alkaline phosphatase-conjugated anti-T and anti-pp53 monoclonal antibodies. The results from these experiments indicate that the T antigen-to-pp53 ratio in the complex is 0.87 +/- 0.27. No statistically significant differences were found in this ratio for faster- and slower-sedimenting complexes. These results from enzyme-linked immunosorbent assays and previous molecular weight estimates of the complex suggest that this complex is composed, on the average, of four molecules of T antigen and four or five molecules of pp53.     

Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.     

Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants

We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated.