Excision of integrated simian virus 40 DNA involving homologous recombination between viral DNA sequences

We have investigated the structure of simian virus 40 (SV40) DNA integrated into the genome of transformed mouse mKS-A cells. We have identified at least six independent integration units containing intact or truncated SV40 DNA sequences. One integration unit was isolated from a genomic mKS-A cell library and investigated by restriction enzyme analysis and partial nucleotide sequencing. This integration unit contains one apparently intact SV40 genome flanked on both sides by truncated versions of the SV40 genome. One of the flanking elements contains a large deletion in the SV40 "late" region and an abbreviated SV40 "early" region. This element was efficiently excised and mobilized after fusion of mKS-A to COS cells. The excision products invariably included the entire SV40 early region even though they were derived from an integrated element lacking this part of the SV40 genome. An analysis of this discrepancy led to the conclusion that the early region sequences were acquired by homologous recombination and, furthermore, that homologous excisional recombination was clearly preferred over non-homologous recombination.     

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

DNA synthesis by partially purified replicating simian virus 40 chromosomes.

We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.     

Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.

Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.     

Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates

The fraction and DNA composition of simian virus 40 chromosomes that were complexed with large T-antigens (T-Ag) were determined at the peak of viral DNA replication. Simian virus 40 chromatin containing radiolabeled DNA was extracted by the hypotonic method of Su and DePamphilis (Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470, 1976) and then fractionated by sucrose gradient sedimentation into replicating (90S) and mature (70S) chromosomes. Viral chromosomes containing T-Ag were isolated by immunoprecipitation with saturating amounts of either an anti-T-Ag monoclonal antibody or an anti—T-Ag hamster serum under conditions that specifically precipitated T-Ag protein from cytosol extracts. An average of 10% of the uniformly labeled DNA in the 90S pool and 7.5% in the 70S pool was specifically precipitated, demonstrating that under these conditions immunologically reactive T-Ag was tightly bound to only 8% of the total viral chromosomes. In contrast, simian virus 40 replicating intermediates (RI) represented only 1.2% of the viral DNA, but most of these molecules were associated with T-Ag. At the shortest pulse-labeling periods, an average of 72 ± 18% of the radiolabeled DNA in 90S chromosomes could be immunoprecipitated, and this value rapidly decreased as the labeling period was increased. Electron microscopic analysis of the DNA before and after precipitation revealed that about 55% of the 90S chromosomal RI and 72% of the total RI from both pools were specifically bound to T-Ag. Comparison of the extent of replication with the fraction of RI precipitated revealed a strong selection for early replicating DNA intermediates. Essentially all of the RI in the 70S chromosomes were less than 30% replicated and were precipitated with anti—T-Ag monoclonal antibody or hamster antiserum. An average of 88% of the 90S chromosomal RI which were from 5 to 75% replicated were immunoprecipitated, but the proportion of RI associated with T-Ag rapidly decreased as replication proceeded beyond 70% completion. By the time sibling chromosomes had separated, only 3% of the newly replicated catenated dimers in the 90S pool (<1% of the dimers in both pools) were associated with T-Ag. Measurements of the fraction of radiolabeled DNA in each quarter of the genome confirmed that T-Ag was preferentially associated with newly initiated molecules in which the nascent DNA was nearest the origin of replication. These results are consistent with a specific requirement for the binding of T-Ag to viral chromosomes to initiate DNA replication, and they also demonstrate that T-Ag does not immediately dissociate from chromosomes once replication begins. The biphasic relationship between the fraction of T-Ag—containing RI and the extent of DNA replication suggests either that 1 or 2 molecules of T-Ag remain stably bound until replication is about 70% completed or that 4 to 6 molecules of T-Ag are randomly released from each RI at a uniform rate throughout replication.     

Superhelix density of replicating simian virus 40 DNA molecules

Simian virus 40 replicating DNA molecules were isolated and fractionated according to the extent of replication by isopynic centrifugation in ethidium bromide-CsCl. Electron microscopic examination of the replicating molecules in the presence of ethidium bromide revealed that the sense of the superhelix in replicating molecules is the same as that of simian virus 40 DNA I. Replicating DNA molecules of differing extents of replication were also analyzed by sedimentation in varying concentrations of ethidium bromide. It was observed that the superhelix density of the unreplicated portion of replicating molecules was greater than that of DNA I and that it increased as the degree of replication increased. In contrast with the increase in superhelix density that was related to the extent of replication, all replicating molecules contained a rather constant number (2 to 5) of additional superhelical turns per molecule, irrespective of the extent of replication. This suggests that a region (or regions) of about 20 to 50 nucleotides may exist in a denatured state in replicating molecules, presumably at the replicating forks of the molecule.     

In situ hybridization of repetitive monkey genome sequences isolated from defective simian virus 40 DNA.

The origin of a repetitive monkey DNA sequence that is incorporated into a defective simian virus 40 genome has been studied. A fragment (about 140 base pairs in length) containing essentially all the repetitive monkey DNA present in the defective and few, if any, SV40 sequences can be cleaved from the purified defective DNA by restriction endonucleases Hind(II and III). Radioactive cRNA prepared with the isolated fragment as template was hybridized in situ to African green monkey chromosomes. The results indicate that all or part of the sequence in question occurs at both centromeric and noncentromeric positions in many, but not all, chromosomes. Of the typical 60 chromosomes, between nine and eleven hybridize with the cRNA in noncentromeric regions.     

Allosteric control of simian virus 40 T-antigen binding to viral origin DNA.

Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored.     

Inhibition of simian virus 40 T-antigen expression by cellular differentiation.

Murine 3T3T stem cells transfected with pSV3neo DNA were employed to study the effects of somatic cell differentiation on simian virus 40 (SV40) T-antigen expression. This experimental approach was used because the 3T3T cell line is a well-characterized in vitro adipocyte differentiation system and the pSV3neo plasmid contains the early region of the SV40 genome and a selective marker, G418 resistance. Cell clones containing stably integrated pSV3neo which expressed T antigen were isolated in G418-containing medium. Most of these cell clones differentiated poorly. However, several clones retained the ability to efficiently differentiate into adipocytes, and with these cell clones, it was established that adipocyte differentiation markedly repressed T-antigen expression. The differentiation-specific repression of T-antigen expression did not result from a loss of proliferative potential associated with terminal differentiation, because it was observed in adipocytes that could be restimulated to proliferate. In such cells, restimulation of cell growth induced reactivation of T-antigen expression. Repression of T-antigen expression was also demonstrated during differentiation of SV40 T-antigen-immortalized human keratinocytes. These results establish that the process of cellular differentiation can repress T-antigen expression in at least two distinct biological systems.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Self-annealing of 4 S strands from replicating simian virus 40 DNA

The nascent short strands (4 S) isolated from replicating Simian, virus 40 DNA hybridize specifically with denatured SV40 DNA and self-anneal extensively (70 to 92%) when incubated at 68 °C in 1 m-NaCl. Since complementary genetic sequences are present in the 4 S strands, both growing chains of SV40 DNA appear to be synthesized discontinuously at each replication fork.     

Localization of T-antigen on simian virus 40 minichromosomes by immunoelectron microscopy.

SV40 chromatin extracted from 42 h post-infected cells by a modification of the standard Triton X-100-EDTA procedure and purified on neutral sucrose gradients was partially immunoprecipitable by a specific SV40 T-antigen (T-Ag) antiserum. Electron microscopic observations of spread minichromosomes were made after labelling by the indirect colloidal gold immunological method using monoclonal antibodies specific for the SV40 T-Ag. In 1-2% of morphologically mature minichromosomes the labelling corresponding to tightly bound T-Ag was localized within the nucleosome-free region near one of its borders. Mapping with three single-cut restriction endonucleases: BamHI, EcoRI and BglI localized the labelling near to, or at the origin of, replication. In addition, we observed that the T-Ag specific antibodies were linked to a DNA-bound particle when the region was not masked by a large clump of antibodies. The variable size of this particle led us to suggest that it might be a complex of T-Ag with other proteins.     

Recurring defective variants of simian virus 40 containing monkey DNA segments.

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.     

In vitro DNA synthesis by an alpha-like DNA polymerase bound to replicating simian virus 40 chromosomes.

Simian virus 40 chromosomes carry out replicative DNA synthesis in vitro which is sensitive to aphidicolin and to N-ethylmaleimide, resistant to 2',3'-dideoxythymidine-5'-triphosphate, and proportional to the amount of chromosome-associated alpha-like polymerase. Thus, an alpha-like DNA polymerase (alpha polymerase or delta polymerase) is responsible for in vitro DNA synthesis.     

Functional analysis of a simian virus 40 super T-antigen.

The SV3T3 C120 line of simian virus 40-transformed mouse cells synthesizes no large T-antigen of molecular weight 94,000 but instead a super T-antigen of molecular weight 145,000. In the accompanying paper (Lovett et al., J. Virol. 44:963-973, 1982), we showed that the integrated viral DNA segment SV3T3-20-K contains a perfect, in-phase, tandem duplication of 1.212 kilobases within the large T-antigen coding sequences. Our data suggested that this integrated template encodes mRNAs of 3.9 and 3.6 kilobases, the smaller of which directs the synthesis of the super T-antigen of molecular weight 145,000. We transfected the DNA segment SV3T3-20-K into nonpermissive rat cells and into TK- mouse L cells and analyzed the T-antigens and viral mRNAs in the transfectants; these data prove directly the coding assignments suggested previously. The super T-antigen retained the ability to induce morphological transformation, and may even transform better than the wild-type protein. It also retained the ability to bind to the cell-coded p53 protein. Transfection into permissive CV-1 cells showed that the super T-antigen encoded by SV3T3-20-K was incapable of initiating DNA replication at the viral origin. The duplication in SV3T3-20-K thus defines a mutation which separates the transformation and DNA replication functions of large T-antigen. We discuss why such mutations may be selected in transformed cells.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.     

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.     

Inhibition of simian virus 40 DNA replication by specific modification of T-antigen with oxidized ATP

We covalently bound periodate-oxidized ATP (oATP) to purified simian virus 40 (SV40) large T-antigen and determined the effect of this modification on viral DNA replication and three other biochemical activities of T-antigen. The oATP bound specifically to T-antigen, inhibiting the ATPase activity and preventing T-antigen from activating SV40 DNA replication in vitro. In contrast, binding of oATP had no effect on the DNA-binding activity of T-antigen nor on its ability to form a complex with DNA polymerase alpha. These results provide direct biochemical evidence suggesting that the T-antigen ATPase activity is necessary for viral DNA replication.