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 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.     

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 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.     

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.     

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.     

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*.     

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.     

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.     

Quantitative analysis of the binding of simian virus 40 large T antigen to DNA

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.     

Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

The origin-specific DNA-binding domain of simian virus 40 large T antigen was analyzed, and its C-terminal boundary was found to be at or before amino acid 259. This does not include the zinc finger structural motif located at amino acids 302 to 320 (J. M. Berg, Science 232:485-486, 1986). Interestingly, N-terminal fragments of 266 and 272 amino acids and larger displayed dramatically reduced origin-binding activity. In addition, the specific DNA-binding properties of truncated proteins purified from both bacterial and mammalian sources were compared. Truncated T antigens from mammalian cells bound specific DNA fragments more efficiently than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulating the DNA-binding activity of large 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.     

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.     

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.     

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.