Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.     

Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T-p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N-terminal half or truncated by various lengths at the C-terminal end, we found that a region between amino acids 114 and 152 and a C-terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C-terminal end up to amino acid 669 is essential for T-p53 complex formation but not the N-terminus up to amino acid 152.     

Identification of a novel antiapoptotic functional domain in simian virus 40 large T antigen.

The ability of DNA tumor virus proteins to trigger apoptosis in mammalian cells is well established. For example, transgenic expression of a simian virus 40 (SV40) T-antigen N-terminal fragment (N-termTag) is known to induce apoptosis in choroid plexus epithelial cells. SV40 T-antigen-induced apoptosis has generally been considered to be a p53-dependent event because cell death in the brain is greatly diminished in a p53-/- background strain and is abrogated by expression of wild-type (p53-binding) SV40 T antigen. We now show that while N-termTags triggered apoptosis in rat embryo fibroblasts cultured in low serum, expression of full-length T antigens unable to bind p53 [mut(p53-)Tags] protected against apoptosis without causing transformation. One domain essential for blocking apoptosis by T antigen was mapped to amino acids 525 to 541. This domain has >60% homology with a domain of adenovirus type 5 E1B 19K required to prevent E1A-induced apoptosis. In the context of both wild-type T antigen and mut(p53-)Tags, mutation of two conserved amino acids in this region eliminated T antigen's antiapoptotic activity in REF-52 cells. These data suggest that SV40 T antigen contains a novel functional domain involved in preventing apoptosis independently of inactivation of p53.     

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen.

To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1-521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyT1-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens.     

Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations.

The simian virus 40 large T antigen is sufficient to confer on cells multiple transformed cell growth characteristics, including growth to a high cell density, rapid growth in medium containing low serum concentrations, and anchorage-independent growth. We showed previously that distinct regions of the protein were involved in conferring these properties and that removal of the first 127 amino acids of T antigen abrogated all three activities. At least three large-T-antigen transformation-related activities have been localized to that region: binding of the tumor suppressor gene product Rb and two independent activities contained within the common region shared by large T and small t antigens. The experiments described here were directed toward determining whether these were the only activities from the N terminus that were needed. To do so we reintroduced an Rb-binding region into the N-terminally truncated T antigen (T128-708) and examined the growth properties of cells immortalized by it in the presence and absence of small t antigen, which can provide the T-common-region transformation-related activities in trans. We show that an Rb-binding region consisting of amino acids 101 to 118, when introduced into a heterologous site in T128-708, is capable of physically binding Rb and that binding is sufficient for cells expressing the protein to acquire the ability to grow to a high saturation density. However, in low-serum medium, the growth rate of the cells and maximal cell density are reduced relative to those of wild-type-T-antigen-expressing cells, and the cells cannot divide without anchorage. This result suggests that although Rb binding is sufficient in the context of T128-708 to confer growth to a high density, one or more other N-terminally located T-antigen activities are needed for cells to acquire the additional growth properties. Small t antigen in trans supplied those activities. These results indicate that the T-common-region activities and Rb binding are the only activities from the T-antigen N terminus needed to restore full transforming activity to the N-terminally truncated T antigen.     

Simian Virus 40 Large T Antigen J Domain and Rb-Binding Motif Are Sufficient To Block Apoptosis Induced by Growth Factor Withdrawal in a Neural Stem Cell Line

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.     

Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties.

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.     

Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.

To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.     

Characterization of an immortalizing N-terminal domain of polyomavirus large T antigen.

Polyomavirus large T antigen has an N-terminal domain of approximately 260 amino acids which can immortalize primary cells but lacks sequences known to be required for DNA binding and replication. Treatment of full-length large T with either V8 protease or chymotrypsin yields an N-terminal fragment of 36 to 40 kDa and a C-terminal fragment of approximately 60 kDa. This finding suggests a division of the protein into two domains. Proteolysis experiments show that the N-terminal domain does not have strong physical association with the rest of the protein. It also does not self-associate. A construct expressing only the N-terminal 259 amino acids is sufficient for immortalization. The independently expressed N-terminal domain is multiply phosphorylated, although at a lower level than the same region in full-length large T. The 259-residue protein binds to both pRb and p107 with somewhat lower efficiency than the full-length protein.     

A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.     

T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123.

Simian virus 40 (SV40) DNA replication begins after two large T-antigen hexamers assemble on the viral minimal origin of replication and locally unwind the template DNA. The activity of T antigen in this reaction is regulated by its phosphorylation state. A form of casein kinase I purified from HeLa nuclear extracts (T-antigen kinase) phosphorylates T antigen on physiologic sites and inhibits its activity in the unwinding reaction (A. Cegielska and D. M. Virshup, Mol. Cell. Biol. 13:1202-1211, 1993). Using a series of mutant T antigens expressed by recombinant baculoviruses in Sf9 cells, we find that the origin unwinding activities of both TS677-->A and TS677,679-->A are inhibited by the T-antigen kinase, as is wild-type T antigen. In contrast, mutants TS120-->A and TS123,679-->A are resistant to inhibition by the kinase. Thus, phosphorylation of serines 120 and 123 is necessary for inhibition of T-antigen activity. Previous studies of casein kinase I substrate specificity have suggested that acidic residues or a phosphorylated amino acid amino terminal to the target residue are required to create a casein kinase I recognition site. However, we find that the T-antigen kinase can add more than 3 mol of Pi per mol to full-length bacterially produced T antigen and that it inhibits the unwinding activity of p34cdc2-activated bacterially produced T antigen. Since no prior phosphorylation is present in this bacterially produced T antigen, and no acidic residues are present immediately amino terminal to serines 120 and 123, other structural elements of T antigen must contribute to the recognition signals for T-antigen kinase. In support of this conclusion, we find that while T-antigen kinase phosphorylates amino-terminal residues in bacterially produced full-length T antigen, it cannot phosphorylate bacterially produced truncated T antigen containing amino acids 1 to 259, a 17-kDa amino-terminal tryptic fragment of T antigen, nor can it phosphorylate denatured T antigen. These findings strongly suggest that the carboxy-terminal domain of T antigen is an important modifier of the recognition signals for phosphorylation of the critical amino-terminal sites by the T-antigen kinase. This conclusion is consistent with previous studies suggesting close apposition of amino- and carboxy-terminal domains of T antigen in the native protein. The three-dimensional conformation of the substrate appears to make a significant contribution to T-antigen kinase substrate specificity.     

The phosphorylation at Thr 124 of simian virus 40 large T antigen is crucial for its oligomerization

SV40 large T antigen is phosphorylated at up to ten different amino acids clustered in an N-terminal and a C-terminal part of the polypeptide chain. The N-terminal phosphorylated residues include Ser 123 and Thr 124. We have analyzed the oligomerization, the complex formation with the cellular oncoprotein p53 and the DNA-binding properties of T antigen from two different SV40 transformed cell lines which have either an amino acid exchange at Ser 123 to Phe (W7) or Thr 124 to Ile (D29). In comparison to wild-type T antigen both mutant T antigens have a slightly reduced binding affinity for both binding sites, I and II, of SV40 DNA. Phosphorylation at both residues of T antigen is not essential for formation of the complex with p53. Only the phosphorylation at Thr 124 seems to be critical for the formation of high molecular mass oligomers. Our data support the hypothesis that the oligomerization of T antigen seems to be implicated in viral DNA replication.     

Phosphorylation pattern of large T antigens in mouse cells infected by simian virus 40 wild type or deletion mutants

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been extensively studied in productive infection of monkey cells. In this study, we analyzed the phosphorylation sites of large T antigen from SV40-infected nonpermissive mouse cells by partial proteolysis fingerprints and analysis of the phosphoamino acids present in the resulting fragments. The wild-type virus and deletion mutants (dl1263, dl1265, dl2194, and dl2198) were used for infection. On the basis of our results and published data (M. Schwyzer, R. Weil, and H. Zuber, J. Biol. Chem. 225:5627-5634, 1980), a cleavage map of large T antigen was established. It was reported that at least four sites of phosphorylation were present. The amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the prolinerich C-terminal end of the molecule at position 701 or 708. On the basis of the concensus as to the amino acid sequence surrounding the recognition sites for protein kinases, it was possible to more precisely locate this phosphothreonine at residue 701. Moreover, the C-terminal part of the molecule contained phosphoserine at a more internal position. In addition, this study firmly established the presence of a phosphothreonine in the N-terminal part of large T antigen. In conclusion, it was shown that the location of phosphorylation sites of large T antigen produced by nonpermissive mouse cells infected by SV40 is strikingly similar to that reported by other groups for large T antigen produced by SV40-infected permissive cells.     

Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants.

The existence of two distinct antigenic sites at the surface of simian virus 40 (SV40)-transformed H-2b cells has been previously demonstrated (A. E. Campbell, L. F. Foley, and S. S. Tevethia, J. Immunol. 130:490-492, 1983) by using two independently isolated SV40-specific cytotoxic T-lymphocyte (CTL) clones, K11 and K19. We identified amino acids in the amino-terminal half of SV40 T antigen that are essential for the recognition of antigenic sites by these CTL clones by using H-2b cells transformed by mutants that produce T antigen truncated from the amino-terminal or carboxy-terminal end or carrying overlapping internal deletions in the amino-terminal regions of SV40 T antigen. The results show that CTL clone K11 failed to recognize and lyse target cells missing SV40 T-antigen amino acids 189 to 211, whereas CTL clone K19 lysed these cells. The cell lines missing SV40 T-antigen amino acids 220 to 223 and 220 to 228 were not lysed by CTL clone K19 but were susceptible to lysis by CTL clone K11. Two other cell lines missing amino acids 189 to 223 and 189 to 228 of SV40 T antigen were not lysed by either of the CTL clones but were lysed by SV40-specific bulk-culture CTL if sufficient amounts of relevant restriction elements were expressed at the cell surface. The SV40 T-antigen amino acids critical for the recognition of an antigenic site by CTL clone K11 were identified to be 193 to 211; 220 to 223 were identified as critical for recognition by CTL clone K19. The deletion of these amino acids from the T antigen resulted in the loss of antigenic sites specific for CTL clones K11 and K19.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

We constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).