Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.     

Unwinding of Parental Strands During Simian Virus 40 DNA Replication

Pools of young (less than 60% replicated) and mature (60-90% replicated) replicating molecules of simian virus 40 (SV40) DNA have been treated at pH 12.2 in order to dissociate growing chains from the parental strands. The molecules are neutralized so that the parental strands can reassociate and they have then been isolated. They are covalently closed structures which sediment rapidly in alkaline sucrose gradients; however, the sedimentation rates are less than the sedimentation rate of SV40 DNA I. Isopycnic banding in CsCl-ethidium bromide and sedimentation velocity studies in the presence of various amounts of ethidium bromide indicate that these structures contain negative superhelical turns and several-fold-higher superhelix densities than SV40 DNA I (the covalently closed DNA molecule). These structures are those that would be predicted if nicking, unwinding, and sealing of the parental strands occurred as replication proceeded. These experiments provide a direct demonstration that there is a progressive decrease in the topological winding number which accompanies SV40 DNA replication.     

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.     

Origin and Direction of Simian Virus 40 Deoxyribonucleic Acid Replication

Double-branched, circular, replicating deoxyribonucleic acid (DNA) molecules of simian virus 40 (SV40) have been cleaved by the R(1) restriction endonuclease from Escherichia coli. This enzyme introduces one double-strand break in SV40 DNA, at a specific site. The site of cleavage in the replicating molecules was used in this study to position the origin and the two branch points. Radioactively labeled molecules fractionated according to their extent of replication were evaluated after cleavage by sedimentation analysis and electron microscopy. The results demonstrate that the R(1) cleavage site is 33% of the genome length from the origin of replication and that both branch points are growing points. These data indicate that SV40 DNA replication is bidirectional and confirm other reports which have shown a unique origin of replication.     

Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

The initiation of simian virus 40 (SV40) DNA replication by the viral T antigen is a complex series of events that begins when T antigen binds specifically to a palindromic arrangement of four GAGGC pentanucleotide sequences in the minimal origin of viral DNA replication (recently reviewed in references 1, 2, 3, 22, and 48). In the presence of Mg-ATP, T antigen assembles cooperatively on the two halves of the palindrome as a double hexamer (10, 11, 13, 24, 30, 38, 51, 53). The DNA conformation flanking the T-antigen binding sites is locally distorted upon hexamer assembly (reference 7 and references therein). One pair of pentanucleotides is sufficient to direct double-hexamer assembly and local distortion of the origin DNA but not to initiate DNA replication (25). ATP hydrolysis by T-antigen hexamers then catalyzes bidirectional unwinding of the parental DNA (reference 53 and references therein). A mutant origin with a single nucleotide insertion in the center of the palindromic T-antigen binding site prevents cooperative interactions between hexamers and cannot support bidirectional origin unwinding (8, 51), suggesting that both processes require interactions between T-antigen hexamers. After assembly of the two replication forks, bidirectional replication is carried out by 10 cellular proteins and T antigen, which remains at the forks as the only essential helicase (reviewed in references 3, 22, and 48).The phosphorylation state of SV40 T antigen governs its ability to initiate viral DNA replication (reviewed in references 15 to 17 and 39). T antigen contains two clusters of phosphorylation sites located at the N and C termini (40, 41). Phosphorylation of T antigen on threonine 124 in the N-terminal cluster was shown to be essential for viral DNA replication in monkey cells and in vitro (5, 14, 32–36, 44). Efforts to define what step in viral DNA replication requires modification of threonine 124 revealed that Mg-ATP-induced hexamer formation of T antigen in solution and DNA helicase activity of T antigen did not require phosphorylation at this site (33, 36). Origin DNA binding of T antigen lacking the modification at residue 124 was weaker than that of the modified T antigen (33, 34, 36, 44), but the reduction in binding was modest under the conditions used for SV40 DNA replication in vitro (36). Moreover, a mutant T antigen containing alanine in place of the phosphorylated threonine (T124A) assembled as a double hexamer on the viral origin and altered the conformation of the early palindrome and AT-rich sequences flanking the T-antigen binding sites in the viral origin in the same manner as the wild-type protein, except that higher concentrations were required (36). However, even at an elevated concentration, these mutant double hexamers were unable to unwind closed circular duplex DNA containing the viral origin (33, 36), suggesting that the defect in unwinding was responsible for the inability of T124A T antigen to replicate SV40 DNA. One possible explanation for the unwinding defect of the mutant T antigen, despite its helicase activity, was that some essential interaction between the two hexamers during bidirectional unwinding depended upon phosphorylation of threonine 124. Electron micrographs of SV40 DNA unwinding intermediates, which showed two single-stranded DNA loops protruding between two hexamers of T antigen, provided support for this explanation, implying that a double hexamer pulled the parental duplex DNA into the protein complex and spooled the single-stranded DNA out (53). Furthermore, double-hexamer formation significantly enhanced the helicase activity of T antigen (47, 47a).Most of the T antigen isolated from mammalian cells is in a hyperphosphorylated form, containing multiple phosphoserines, as well as two phosphothreonines, and supports SV40 DNA replication in vitro poorly but can be stimulated by treatment with alkaline phosphatase or protein phosphatase 2A (19, 28, 37, 42, 49, 50). Hyperphosphorylated T antigen is unable to unwind duplex closed circular duplex DNA harboring the viral origin (4, 6, 51). Dephosphorylation of serines 120 and 123 restores its ability to unwind origin DNA (14, 43, 51). Studies of double-hexamer assembly on the origin indicate that phosphorylation of T antigen on serines 120 and 123 also impairs the cooperativity of double-hexamer assembly (14, 51). These results demonstrate that hyperphosphorylation of T antigen interferes with interactions between hexamers that are required for origin unwinding and raise the question of whether the phosphorylation state of threonine 124 might also affect the cooperativity of double-hexamer assembly on the viral origin.One class of T antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has been reported to display properties similar to those of the T124A mutant and the hyperphosphorylated form of T antigen (54). Class 4 mutant proteins are defective in viral DNA replication in vivo and in vitro, bind to the viral origin as double hexamers and alter the local DNA conformation, and have helicase activity but do not unwind closed circular duplex viral DNA. The replication and unwinding defects could be due to faulty phosphorylation patterns or to other malfunctions not dependent on phosphorylation status.The work presented here was undertaken to reevaluate the assembly of wild-type and T124A T antigen on SV40 origin DNA by using more-sensitive quantitative assays and to compare them with the class 4 mutants. We report that cooperativity of T124A T antigen in double-hexamer assembly on the viral origin is impaired. The class 4 mutant T antigens were also found to have defects in cooperativity of double-hexamer assembly. T124A T antigen inhibited the ability of the wild-type protein to unwind closed circular duplex origin DNA. Both T124A and the class 4 mutants displayed dominant-negative phenotypes in viral DNA replication in vitro. Based on these observations, we propose that the N-terminal cluster of phosphorylation sites and the DNA binding domain mediate cooperative hexamer-hexamer interactions during assembly on the viral origin and speculate that these regions of T antigen may interact during origin DNA unwinding.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

Loss of Polyoma Virus Infectivity as a Result of a Single Amino Acid Change in a Region of Polyoma Virus Large T-Antigen Which Has Extensive Amino Acid Homology with Simian Virus 40 Large T-Antigen

The polyoma virus (Py) transformed cell line 7axB, selected by in vivo passage of an in vitro transformed cell, contains an integrated tandem array of 2.4 genomes and produces the large, middle, and small Py T-antigen species, with molecular weights of 100,000, 55,000, and 22,000, respectively (Hayday et al., J. Virol. 44:67-77, 1982; Lania et al., Cold Spring Harbor Symp. Quant. Biol. 44:597-603, 1980). The integrated viral and adjacent host DNA sequences have been molecularly cloned as three EcoRI fragments (Hayday et al.). One of these fragments (7B-M), derived from within the tandem viral sequences, is equivalent to an EcoRI viral linear molecule. Fragment 7B-M has been found to be transformation competent but incapable of producing infectious virus after DNA transfection (Hayday et al.). By constructing chimerae between 7B-M and Py DNA and by direct DNA sequencing, the mutation responsible for the loss of infectivity has been located to a single base change (adenine to guanine) at nucleotide 2503. This results in a conversion of an aspartic acid to a glycine in the C-terminal region of the Py large T-antigen but does not appear to affect the binding of the Py large T-antigen to Py DNA at the putative DNA replication and autoregulation binding sites. The mutation is located within a 21-amino acid homology region shared by the simian virus 40 large T-antigen (Friedmann et al., Cell 17:715-724, 1979). These results suggest that the mutation in the 7axB large T-antigen may be involved in the active site of the protein for DNA replication.     

Relationship Between Replication of Simian Virus 40 DNA and Specific Events of the Host Cell Cycle

The relationship between replication of simian virus 40 (SV40) DNA and the various periods of the host-cell cycle was investigated in synchronized CV(1) cells. Cells synchronized through a double excess thymidine procedure were infected with SV40 at the beginning or the middle of S, or in G(2). The first viral progeny DNA molecules were in all instances detected approximately 20 h after release from the thymidine block, independent of the time of infection. The length of the early, prereplicative phase of the virus growth cycle therefore depended upon the period of the cell cycle at which the cells were infected. Infection with SV40 was also performed on cells obtained in early G(1) through selective detachment of cells in metaphase. As long as the cells were in G(1) at the time of infection, the first viral progeny DNA molecules were detected during the S period immediately following, whereas if infection took place once the cells had entered S, no progeny DNA molecule could be detected until the S period of the next cell cycle. These results suggest that the infected cell has to pass through a critical stage situated in late G(1) or early S before SV40 DNA replication can eventually be initiated.     

Sequence Requirements for the Assembly of Simian Virus 40 T Antigen and the T-Antigen Origin Binding Domain on the Viral Core Origin of Replication

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.     

Susceptibility of Human Cell Strains to Transformation by Simian Virus 40 and Simian Virus 40 Deoxyribonucleic Acid

Marked differences were found in the susceptibility of human fibroblasts to transformation by simian virus 40 (SV40). Highly susceptible cell strains were derived from patients with diseases associated with chromosomal abnormalities and a high incidence of tumors. In the present study, SV40 transformation-susceptible cell strains were not found to have a generalized increase in viral sensitivity. The differences in transformation frequency among cell strains with whole virus are eliminated by the use of isolated SV40 deoxyribonucleic acid, suggesting that the relative resistance of most cell strains to transformation by whole virus is due to a block at an early step in infection.     

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.     

Replication Stress and Mitotic Dysfunction in Cells Expressing Simian Virus 40 Large T Antigen

We previously demonstrated that simian virus 40 (SV40) large T antigen (LT) binds to the Bub1 kinase, a key regulator of the spindle checkpoint and chromosome segregation. Bub1 mutations or altered expression patterns are linked to chromosome missegregation and are considered to be a driving force in some human cancers. Here we report that LT, dependent on Bub1 binding, causes micronuclei, lagging chromatin, and anaphase bridges, which are hallmarks of chromosomal instability (CIN) and Bub1 insufficiency. Using time-lapse microscopy, we demonstrate that LT imposes a Bub1 binding-dependent delay in the metaphase-to-anaphase transition. Kinetochore fibers reveal that LT, via Bub1 binding, causes aberrant kinetochore (KT)-microtubule (MT) attachments and a shortened interkinetochore distance, consistent with a lack of tension. Previously, we showed that LT also induces the DNA damage response (DDR) via Bub1 binding. Using inducible LT cell lines, we show that an activated DDR was observed before the appearance of anaphase bridges and micronuclei. Furthermore, LT induction in serum-starved cells demonstrated γ-H2AX accumulation in cells that had not yet entered mitosis. Thus, DDR activation can occur independently of chromosome segregation defects. Replication stress pathways may be responsible, because signatures of replication stress were observed, which were attenuated by exogenous supplementation with nucleosides. Our observations allow us to propose a model that explains and integrates the diverse manifestations of genomic instability induced by LT.     

Initiation Points for DNA Replication in Nontransformed and Simian Virus 40-Transformed BALB/c 3T3 Cells

The number of initiation points for DNA synthesis per unit length of DNA in rapidly growing cells is greater for simian virus 40-transformed than for nontransformed BALB/c 3T3 cells.     

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear.     

Viral DNA Synthesis in Cells Infected by Temperature-Sensitive Mutants of Simian Virus 40

Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.     

Hypoxia Blocks In Vivo Initiation of Simian Virus 40 Replication at a Stage Preceding Origin Unwinding

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia show a burst of viral replication immediately after reoxygenation. DNA precursor incorporation and analysis of growing daughter strands by alkaline sedimentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replication round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation. A distinct form of circular closed, supercoiled SV40 DNA was detectable as soon as 3 min after reoxygenation but not under hypoxia. Sensitivity to the DNA nuclease Bal 31 and migration behavior in chloroquine-containing agarose gels suggested that this DNA species was highly underwound compared to other SV40 topoisomers and was probably related to the highly underwound form U DNA first described by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 84:16–20, 1987), in vitro. 3′-OH ends of presumed RNA-DNA primers could be detected in form U by 3′ end labeling with T7 polymerase. Addition of aphidicolin to the cells before reoxygenation led to a pronounced accumulation of form U DNA containing RNA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidicolin-treated SV40-infected cells showed that form U is an initial intermediate of SV40 DNA replication which matures into higher-molecular-weight replication intermediates and into SV40 form I DNA after removal of the inhibitor. These results suggest that in vivo initiation of SV40 replication is arrested by hypoxia before origin unwinding and primer synthesis.     

Vaccination with Attenuated Simian Immunodeficiency Virus by DNA Inoculation

Delivering attenuated lentivirus vaccines as proviral DNA would be simple and inexpensive. Inoculation of macaques with wild-type simian immunodeficiency virus strain mac239 (SIV(mac239)) DNA or SIV(mac239) DNA containing a single deletion in the 3' nef-long terminal repeat overlap region (nef/LTR) led to sustained SIV infections and AIDS. Injection of SIV(mac239) DNA containing identical deletions in both the 5' LTR and 3' nef/LTR resulted in attenuated SIV infections and substantial protection against subsequent mucosal SIV(mac251) challenge.