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.     

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

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.     

Independent expression of the transforming amino-terminal domain of SV40 large I antigen from an alternatively spliced third SV40 early mRNA.

We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as well as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat F111 fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain.     

Comparative study of papovavirus DNA: BKV(MM), BKV(WT) and SV40.

Extensive physical mapping revealed that approximately 90% of the genomes of BKV(prototype, WT) and BKV (MM strain) are identical or closely related. Nucleotide sequences of the non-homologous regions and a large portion of the homologous regions have been determined for both genomes. The coding sequence of small t antigen of BKV(MM) is 216 nucleotides shorter than that of BKV(WT), even though no differences in biological function of the t antigen was observed. Both genomes contain three similar sets of 44-61 base-pair repeated sequences. However, the DNA sequence of the tandem repeats is totally different between BKV (human cell as host) and SV40 (monkey cell as host). On the other hand, the region between the N-terminus of the T antigen genes and the origin of replication is dominated by a similar set of palindromic sequences in BKV and SV40 DNA. There is also extensive homology between the regions which code for proteins in BKV and SV40, suggesting a close evolutionary relationship.     

The Origin DNA-Binding and Single-Stranded DNA-Binding Domains of Simian Virus 40 Large T Antigen Are Distinct

Little is known about the ability of simian virus 40 (SV40) T antigen to bind single-stranded DNA. We demonstrate here that a mutant (259-708) missing the first 258 amino acids of T antigen and its origin-binding domain bound single-stranded DNA at close to normal levels, whereas a mutant containing only the first 259 amino acids failed to bind any single-stranded DNA. The 259-708 mutant also assembled into high-molecular-weight oligomers in the presence of single-stranded DNA. Its ATPase activity was stimulated by single-stranded DNA similarly to the wild type (WT). Furthermore, WT T antigen’s ability to bind to single-stranded DNA was inhibited by the binding of two monoclonal antibodies that recognize a region after residue 362. These results show that the domain responsible for binding to single-stranded DNA is completely separate from the origin-binding domain.     

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.     

Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124.

Previous studies have shown that phosphorylation of simian virus 40 (SV40) T antigen at threonine 124 enhances the binding of T antigen to the SV40 core origin of replication and the unwinding of the core origin DNA via hexamer-hexamer interactions. Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication. Phosphorylation, therefore, activates the minimal DNA binding domain of T antigen even in the absence of domains required for hexamer formation. Activation is mediated by only one of three DNA binding elements in the minimal DNA binding domain of T antigen. This element, including amino acids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found in ancillary regions of the SV40 origin of replication. Interestingly, the same four pentanucleotides in the core origin are necessary and sufficient for phosphorylation-enhanced DNA binding. Further, we show that phosphorylation of threonine 124 promotes the assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA. We propose that phosphorylation induces conformational shifts in the minimal DNA binding domain of T antigen and thereby enhances interactions among T-antigen subunits oriented by core origin pentanucleotides. Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer complexes and origin unwinding.     

Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli

The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.     

BK virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology.

The DNA sequence of the early region of the human papovavirus BK (MM strain) was determined. A potential initiation signal for translation is located at nucleotides 3,047 to 3,045 or map position 0.614. Extending counterclockwise from this AUG signal there is only one open reading frame, which can code for a putative t antigen of 100 amino acids in length. If the early mRNA of BKV is spliced, then the regions between nucleotides 3,047 to 2,808 and 2,725 to 884 can code for a T antigen 694 amino acids in length. The sequences of the deduced T antigens in BK virus share 71% amino acid homology with those in simian virus 40, whereas the coding sequences of the two viruses share 70% DNA homology. Comparison of DNA sequences and evaluation of homology measurements between these two viruses are discussed.     

A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions.

Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.     

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.     

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation.

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells.     

Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens.

The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.     

Human Papovavirus, BK Strain: Biological Studies Including Antigenic Relationship to Simian Virus 40

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.     

The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus

The association between the retinoblastoma protein (p105-RB) and either the large T antigen of SV40 or the E1A proteins of adenovirus is thought to be an important step in transformation by these viral oncogenes. E1A and large T antigen share a small region of amino acid homology that is necessary for high affinity binding with p105-RB. Mutations of this homology region were shown to reduce drastically the frequency of transformation mediated by the E1A or large T oncogenes. Previously, this small region in E1A was shown to be sufficient for interaction with a second cellular protein of 107,000 daltons (107K). Here we show that in human cells, the large T antigens of SV40 or JC virus also form complexes with 107K. Demonstration of complexes between 107K and the large T antigens of SV40 and JC virus suggests that these associations may represent another component of a common mechanism for transformation between adenoviruses and polyoma viruses.