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.     

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.     

The simian virus 40 small-t and large-T antigens jointly regulate cell cycle reentry in human fibroblasts

Focus formation in human diploid fibroblasts (HDF cells) is known to require both the simian virus 40 (SV40) large-T and small-t antigens. Similarly, both SV40 proteins were required to stimulate confluent, density-arrested HDF cells to reenter the cell cycle. This study used defective recombinant adenoviruses to examine the roles of the individual SV40 proteins in altering specific steps in the cell cycle. Small-t antigen and, to a lesser extent, large-T antigen increased the level of the S phase cyclin cyclin A but without increasing the activity of associated cyclin kinases unless the two SV40 proteins were coexpressed. The absence of kinase activity reflected the presence in density-arrested cells of high levels of the cyclin-dependent kinase inhibitors p21(WAF1) and p27(KIP1). We report here that expression of SV40 large-T antigen reduced levels of p21(WAF1), while expression of small-t antigen was required to decrease p27(KIP1). The separate effects of large-T and small-t antigens on these two inhibitors may explain the joint requirement for the two proteins to drive cell cycle reentry of HDF cells and ultimately transform these cells.     

Mutational analysis of simian virus 40 small-t antigen.

Several point mutations in the simian virus 40 (SV40) small-t antigen have been analyzed for their effects on protein stability, transformation, transactivation, and binding of two cellular proteins. All mutations which affected cysteine residues in two cysteine clusters produced highly unstable small-t antigens. Four point mutations outside these clusters and one in-frame deletion mutant, dl890, produced stable proteins but reduced transformation efficiency. These were able to transactivate the EII promoter and bind the cellular proteins, suggesting that these activities are not sufficient for small-t-mediated enhancement of transformation.     

Control of protein phosphatase 2A by simian virus 40 small-t antigen.

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.     

Characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-T antigens.

Simian virus 40 small-t and large-T antigen were synthesized in vitro and labeled with methionine donated by initiator tRNA. Tryptic peptide fingerprinting was used to identify the amino-terminal peptide of the two proteins. Similar fingerprint analysis of small-t and large-T made in vitro in the absence of acetyl coenzyme A showed that the mobility of the amino-terminal peptide was changed under these conditions and suggested that it is acetylated. These data establish that the amino-terminal methionine residue of simian virus 40 small-t and large-T results from an initiation event, not post-translational cleavage, and provides additional evidence that the amino terminus of both proteins is acetylated. The identification of the amino-terminal peptide provides a useful marker for further studies on different forms of T-antigen from cells infected with and transformed by simian virus 40 and related viruses.     

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.     

Nucleotide sequence deletions within the coding region for small-t antigen of simian virus 40.

Simian virus 40 early mutants with deletions mapping in the 0.53-0.60 region have been sequenced by the Maxam and Gilbert approach. All these deletions effect the small-t gene. The size of the shortened small-t-related polypeptides produced by several of the mutants has been compared with the molecular weight as deduced from the nucleotide sequence. There was good agreement for the mutants dl890, dl891, and dl2102. For dl2121 and dl2122 the small-t-related protein was considerably larger than expected. It is possible to explain this result on the basis of the nucleotide sequence: the normal splicing event of the small-t mRNA still occurs, but as the deletion shifts the reading frame, translation of the small-t-related polypeptide continues beyond the small-t splice, but in a different reading frame than large-T. Mutants dl883, dl884, and dl2112 have lost one of the small-t splicing boundaries, and no (or minute amonts of) small-t-related protein has been observed in mutant-infected cells. The possible relationship between splicing and transport of polyadenylic acid-containing mRNA from the nucleus to the cytoplasm in vertebrae cells is discussed.     

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Nucleotide sequence of the simian virus 40 HindII + III restriction fragment A (second part of the T antigen gene)

The nucleotide sequence of the second part of the simian virus 40 DNA HindII + III restriction fragment A is presented. The sequence extends from map position 0.533 to 0.424 and together with the first part of Hind-A [Volckaert et al. Proc. Natl Acad. Sci. U.S.A. 75, 2160--2164 (1978)] completes the total hind-A sequence, comprising 1169 base pairs. The second half of Hind-A includes the region corresponding to the second splicing boundary common to small tumor antigen (small-t) and large tumor antigen (large-T) mRNA and it contains coding information for an internal portion of large-T antigen. Two similar secondary structures of reasonable thermodynamic stability can be proposed for the nucleotide sequence of the pre-mRNA corresponding to the region reported here. Their possible relevance to the splicing of the SV40 early mRNAs is discussed. The deduced amino acid sequence is 188 residues long and contains a Lys-Lys-Lys-Arg-Lys-stretch which may be involved in the DNA binding capacity of large-T. A presumptive phosphorylation site is also present.     

Characterization of two SV40 early mRNAs and evidence for a nuclear "prespliced" RNA species.

Using in vitro translation of sucrose-gradient fractionated cytoplasmic mRNA from SV40-infected cells, we have shown that a deletion in the region mapping between 0.54--0.59 reduced the size of mRNA for small-t but not the size of mRNA for large-T. Mutants with a deletion in this region were shown to produce in vivo either shortened small-t or no small-t, and normal large-T. Similarly, in vitro translation of poly(A)+cytoplasmic RNA from cells infected with these mutants gave the same results. On the other hand in vitro translation of poly(A)+nuclear RNA from the mutants which made no small-t produced a small-t derivative possibly synthesized from a prespliced RNA species. We have also shown that poly(A)+nuclear RNA from mutant dl 2122 produced two small-t related proteins: one of these (MW: 11K) probably represents the product of a "prespliced" RNA, the other (MW: 17K) which is also found in the cytoplasm represents the product of the mutant specific small-t mRNA.     

Extraction and fingerprint analysis of simian virus 40 large and small T-antigens.

A study of simian virus 40 (SV40) T-antigens isolated from productively infected CV1 cells using a variety of different extraction procedures showed that under some conditions the highest molecular weight form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV40-transformed H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent molecular weight in the range 15,000 to 20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV40 proteins showed that small-t has 10 to 12 methionine peptides whereas large-T has 15 to 18 methionine peptides. All but two of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV40 VP1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV40 DNA.     

Hepatic expression of SV40 small-T antigen blocks the in vivo CD95-mediated apoptosis

We have previously demonstrated that CD95-mediated apoptosis of hepatocytes is blocked in a murine model of hepatocarcinogenesis due to the expression of SV40 early sequences encoding the large-T and small-t antigens. In this study, we set out to pinpoint the sequences involved in this apoptosis-resistant phenotype, and tested several mutants of the SV40 early region for their ability to confer protection against CD95-induced apoptosis in transgenic mice. We show that resistance to apoptosis is independent of the transforming character of the mutants and demonstrate that the expression of the small-t antigen alone in transgenic mice is sufficient to confer this resistance. Our data also reveal an increased level of activated Akt kinase in these transgenic mice, and this could account for this hitherto unknown function of the SV40 small-t antigen.     

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.     

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.