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.     

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.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Reassortment of Simian Virus 40 DNA During Serial Undiluted Passage

Alterations occur in the supercoiled form of viral DNA after the serial undiluted passaging of simian virus (SV) 40. We have identified a portion of the viral genome which is amplified during this process. These SV40 DNA sequences represent about 30% of the viral genetic information and are present in a reiterated form in twisted circular molecules prepared from purified virions. In addition, reiterated and unique green monkey DNA sequences are incorporated into supercoiled viral DNA. The cellular DNA appears to be inserted at numerous locations in the DNA I molecules.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

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.     

Specific Initiation Site for Simian Virus 40 Deoxyribonucleic Acid Replication

Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with (3)H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication.     

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.     

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.     

Simian Virus 40-Host Cell Interaction During Lytic Infection

Both exponentially growing and serum-arrested subcloned CV-1 cell cultures were infected with simian virus 40 (SV40). By 24 h after infection 96% of the nuclei of these permissive cells contained SV40 T-antigen. Analysis of the average DNA content per cell at various times after infection indicated that by 24 h most of the cells contained amounts of DNA similar to those normally found in G(2) cells. Analysis of cell cycle distributions indicated that a G(2) DNA complement was maintained by over 90% of the cells in the infected populations 24 to 48 h postinfection. Cells continued to synthesize SV40 DNA during the first 50 h after infection, and cytopathic effect was first observed 60 h after inoculation. After infection the number of mitotic cells that could be recovered by selective detachment decreased precipitously and was drastically reduced by 24 h. A study of the kinetics of decline in the number of mitotic cells suggests that this decline is related to an event during the cell cycle at or near the G(1)-S-phase border upon which commencement of SV40 DNA replication apparently depends. It was concluded that after SV40 infection, stationary cells are induced to cycle, and cycling cells complete one round of cellular DNA synthesis but do not divide. Although the infected cells continue to synthesize viral DNA, they do not appear able to reinitiate cellular DNA replication units. These results imply that the abundance of T-antigen (produced independently of cell cycle phase) in the presence of the enzymes required for continued DNA synthesis is not sufficient for reinitiation of cellular DNA synthesis.     

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.     

Initial Site of Synthesis of Virus During Rescue of Simian Virus 40 from Heterokaryons of Simian Virus 40-Transformed and Susceptible Cells

Simian virus 40 (SV40) can be rescued from certain SV40-transformed hamster cells by fusion with susceptible African green monkey kidney (CV-1) cells, in the presence of ultraviolet-irradiated Sendai virus. We have determined the sites in which SV40 is produced during rescue in these heterokaryons. To determine the sequence, nuclei were isolated from fused cells at various times after fusion, separated on sucrose-density gradients, and assayed for infectious center formation and virus content on CV-1 monolayers. Virus was first detected in the transformed nucleus (40 hr postfusion), and later associated with both transformed and susceptible nuclei (68 to 72 hr). Viral rescue apparently does not depend upon the transfer of SV40 deoxyribonucleic acid to a susceptible CV-1 nucleus, since the transformed nucleus is the primary site of virus production. The time course of certain cytological events in the rescue process and in productive infection was found to be similar.     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

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.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

Dimerization of simian virus 40 T-antigen hexamers (TAg_H) into double hexamers (TAg_DH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAg_DH with DNA during unwinding, we examined the binding of TAg_DH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAg_DH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAg_DH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ~17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAg_H encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAg_H interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.     

Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.