p53 and DNA polymerase alpha compete for binding to SV40 T antigen

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.     

Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication

Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.     

N-Acetoxy-acetylaminofluorene reacts preferentially with a control region of intracellular SV40 chromosome

Many chemical carcinogens or their metabolites react with DNA; thus it is of interest to determine what effect chromosomal structure has on these reactions. The chromosome of simian virus 40 (SV40) is well suited for such studies; like chromatin of eukaryotic cells, it is organized into nucleosomes. The nucleotide sequence of SV40 is known, together with much about the pattern of viral gene expression and DNA replication, and the structure of the viral chromosome. We have investigated the binding of the ultimate carcinogen, N-acetoxy-acetylaminofluorene (AAAF), to specific regions of the SV40 chromosome in situ in the intact infected cell. The results, reported here, indicate that a region containing regulatory functions on the intracellular SV40 chromosome has unique structural properties which render it more susceptible to attack by AAAF than the rest of the SV40 genome. The preferential binding of AAAF to regulatory regions of chromatin may have implications for the mechanism of action of this and similar carcinogens.     

Tightly associated lipids may anchor SV40 large T antigen in plasma membrane

Simian virus 40 (SV40) large T antigen, a multifunctional protein necessary for lytic growth and cell transformation, is located mainly in the nucleus and in small amounts on the cell surface (surface T). Surface T may have a passive role in SV40 tumour rejection by cytotoxic T cells as a component of SV40-TSTA (tumour-specific transplantation antigen). The unusual induction of this immune response by immunizing mice with soluble T antigen led us to investigate the in vitro binding of T antigen to the surface of living cells in more detail. Our results show that native surface T and a minor subset of large T antigen having a high cell surface binding affinity in vitro, behave like integral membrane proteins. Several viral proteins including SV40 T antigen and cellular proteins seem to be linked to fatty acids (acylation). To analyse whether this mechanism is involved in the stable attachment of in vitro-bound T antigen to the plasma membrane of living target cells, we determined the degree of labelling of this molecule by using target cells prelabelled with 3H-fatty acid. Here we report that T antigen extracted from unlabelled SV40-transformed cells (SV80) becomes 3H-labelled after in vitro binding to the cell surface of 3H-palmitate-prelabelled HeLa cells. These results suggest that T antigen attached externally to living cells, may be anchored by tightly linked lipids.     

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.     

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.     

Expression of the chromosomal mouse Beta maj-globin gene cloned in SV40.

The complete chromosomal mouse Beta maj-globin gene, including its intervening and flanking sequences, has been cloned in the monkey virus SV40. the mouse gene is transcribed, processed and translated in infected monkey kidney cells to yield mouse Beta maj-globin.     

A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products

SV40 large T antigen contains a small region of amino acid sequence, conserved among the papovaviruses, that shows considerable similarity to conserved domain 2 of the adenovirus E1A oncogene, a domain which plays an important role in the E1A transforming functions. To learn whether the analogous SV40 T antigen sequences could substitute functionally for E1A domain 2, a chimaeric gene was constructed, coding for T antigen amino acid residues 101 to 118 in place of E1A domain 2. The resulting product showed much of the activity of the wild-type E1A products. It induced proliferation of primary BRK cells and cooperated with the ras oncogene to transform these cells fully. In addition, the chimaeric protein coprecipitated two cellular proteins whose specific binding to the E1A products depends on the presence of domain 2. The activity of the chimaeric product suggests that a similar functional unit exists in the transforming proteins of both SV40 and adenovirus, and that these proteins may exert their cell growth regulating effects through similar mechanisms.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome.

Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.     

Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.     

Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus

The simian virus 40 (SV40) 72-base pair (bp) tandem-repeated sequences have a crucial role as an activator element in viral gene expression. We replaced the SV40 72-bp repeat with a 72-bp repeat derived from the long terminal repeat (LTR) of cloned Moloney murine sarcoma virus (MSV) DNA. Although there is no detectable sequence homology to SV40, the MSV repeats can substitute functionally for the SV40 repeats and generate a viable virus in monkey kidney cells.     

SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumours in transgenic mice

We have shown recently that choroid plexus tumours frequently develop in transgenic mice which have developed from fertilized eggs injected with DNA molecules containing both simian virus 40 (SV40) early-region genes and metallothionein (MT) fusion genes, and several lines of mice have now been established in which all of the offspring that inherit the foreign DNA succumb to these tumours at 3-5 months of age (ref. 1 and our unpublished data). Several other tissues, notably thymus and kidney, occasionally also show pathological changes. SV40 large-T antigen protein and messenger RNA are always present in affected tissues at much greater concentrations than in unaffected tissues, suggesting that SV40 early-region genes are preferentially activated in choroid plexus, thymus and kidney and that this activation frequently leads to tumorigenesis in the choroid plexus. To determine which regions of the original constructs are important for this tumorigenesis, we have now tested several derivatives and report here that the large-T antigen is sufficient, that the MT fusion gene is dispensable and that the SV40 enhancer (72-base-pair repeat region) has an important role in directing tumours to the choroid plexus. Deletion of the SV40 enhancer region alone commonly leads to peripheral neuropathy, as well as liver and pancreatic tumours, which are the subject of the accompanying paper. Evidence is presented that these pathologies may result from an enhancing effect of the MT sequences on large-T antigen genes, made possible by removal of the otherwise dominant SV40 enhancer.     

Mouse p53 inhibits SV40 origin-dependent DNA replication

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.     

The class I major histocompatibility antigen gene activated in a line of SV40-transformed mouse cells is H-2Dd, not Qa/Tla

We have previously described several complementary DNA clones isolated because they correspond to messenger RNAs present at higher levels in the simian virus 40 (SV40)-transformed BALB/c 3T3 cell line SV3T3 Cl38 than in the normal, parental BALB/c 3T3 line. One of these clones, pAG64, hybridizes to RNAs which, while present in BALB/c 3T3 cells, are 10-20-fold more abundant in SV3T3 Cl38 and are found at high levels in a wide variety of transformed cell lines. Nucleotide sequence analysis showed that pAG64 encodes a class I antigen of the major histocompatibility complex. To ascertain the identity of pAG64, we compared its sequence with the available sequences of d haplotype class I antigen genes [K locus, L locus, D locus and the Qa gene defined by genomic clone 27.1] and found that it showed multiple clustered differences from each of these sequences. We therefore concluded that it was not derived from the H-2Kd, H-2Ld or H-2Dd genes and thus must correspond to one of the other class I antigen genes, namely those of the Qa/Tla complex, although it was clearly not the Qa gene defined by the genomic clone 27.1. We now report subsequent findings which indicate that pAG64 in fact corresponds to the H-2Dd gene and not to a Qa/Tla gene.