Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.     

Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) on UV-irradiated monkey kidney CV-1P cells at 33° was increased over survival on unirradiated cells. During this process — called induced-virus reactivation — the progeny virus yielded by UV-irradiated cells had a much higher mutation frequency than did the progeny from unirradiated cells. Mutation rates were quantified by using phenotypic reversion towards wild-type growth of an early (tsA 58) or a late (tsB 201) temperature-sensitive SV40 mutant. Analysis of SV40 revertant genomes indicated that no detectable deletions or additions were resposible for the reversion process.These results suggest that enzymes from UV-irradiated cells are able to replicate UV-irradiated DNA by an error-prone mode of DNA repair. Induced virus reactivation and error-prone replication are probably one of the expressions of SOS functions in mammalian cells.     

Characterization of simian virus 40 transformed African green monkey cells (CV-1). I. Defective virion and viral genome.

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.     

Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.     

UV-reactivation,virus production and mutagenesis of SV40 in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) is higher in UV-irradiated than in non-irradiated monolayers of BSC-1 monkey cells. A similar reactivation is found when cells are infected with SV40-DNA, suggesting that reactivation acts on viral DNA. The enhanced reactivation of UV-irradiated SV40 and SV40-DNA is optimal when infection is delayed for 2–3 days after irradiation of the cells.UV-pretreated cells infected with SV40-DNA produce more virus than infected control cells; the time curve of this process is similar to that found for enhanced virus reactivation and suggests that facilitated virus production in UV-irradiated cells and enhanced virus reactivation might be manifestations of the same process.If the non-irradiated SV40 thermosensitive mutant BC245 is propagated in UV-irradiated BSC-1 cells the rate of back mutation to phenotypically wild-type is increased compared with that of the control. This suggests that an inducible error-prone system is functional in these cells. When the UV-irradiated tsBC245 is propagated in non-irradiated cells the reversion frequency is greatly enhanced, which suggests that either the introduction of UV-irradiated SV40-DNA is sufficient to induce an error-generating system, or that a constitutive error-prone mechanism is operative on this DNA.     

Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.     

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.     

Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin.

Detergent extraction of simian virus 40 (SV40) DNA from infected monkey CV-1 cells, after a brief exposure to the drug camptothecin, yields covalent complexes between topoisomerase I and DNA that band with reduced buoyant densities in CsCl. The following lines of evidence indicate that the enzyme is preferentially associated with SV40 replicative intermediates. First, the percentage of the isolated labeled viral DNA that exhibited a reduced buoyant density is inversely proportional to the length of the labeling period and approximately parallels the percentage of replicative intermediates for each labeling time (5 to 60 min). Second, after labeling for 60 min, the isolated low-density material was found to be enriched for replicative intermediates as measured by sedimentation in neutral sucrose. Third, analysis of extracted viral DNA by equilibrium centrifugation in CsCl-propidium diiodide gradients that separate replicating molecules from completed form I DNA revealed that camptothecin pretreatment specifically caused the linkage of topoisomerase I to replicating molecules. In addition, analysis of the low-density material obtained under conditions when only the newly synthesized strands of the replicative intermediates were labeled showed that the enzyme was associated almost exclusively with the parental strands. Taken together, these observations indicate that topoisomerase I is involved in DNA replication, and they are consistent with the hypothesis that the enzyme provides swivels to allow the helix to unwind. The observed bias in the distribution of topoisomerase I on intracellular SV40 DNA could be the result of rapid encapsidation of replicated molecules that precludes the association of topoisomerase I with the DNA or, alternatively, the result of a specific association of the enzyme with replicative intermediates.     

Identification of Virus-Induced Proteins in Cells Productively Infected with Simian Virus 40

The number and molecular weight of the structural polypeptides of highly purified simian virus 40 (SV40) were determined by polyacrylamide gel electrophoresis. Six different polypeptides were found, two of which (VP1 and VP2) comprise the bulk of the viral capsid proteins. The pattern of protein synthesis in productively infected CV-1 cells was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identification of virus-induced proteins in the infected CV-1 cells was achieved in double-labeling experiments by electrophoresis with purified labeled SV40 capsid proteins. Four of these proteins (VP1 and VP4) could be classified as components of the virion because their synthesis occurred after the onset of viral deoxyribonucleic acid (DNA) replication and because they were inhibited by arabinofuranosylcytosine (ara-C). Appearance of two other virus-induced proteins was not prevented by ara-C; one of them did not comigrate in the electrophoresis with purified virion polypeptides, and both could be detected before the onset of viral DNA synthesis. These latter two proteins were classified on the basis of these criteria as nonvirion capsid proteins (NCVP1 and NCVP2).     

Kinetics of DNA and histone messenger RNA synthesis in CV-1 monkey kidney cells infected with simian virus 40.

Histone messenger RNA has been identified in CV-1 monkey kidney cells and its synthesis during the simian virus 40 (SV40) productive cycle has been correlated with the synthesis of cellular DNA and viral DNA. In cultures of CV-1 cells that have reached confluence, infection with SV40/5 (a high-yield clone of SV40) promotes an increase in the rate of cellular DNA synthesis followed by a decline. During this decline the rate of viral DNA synthesis continues to rise and eventually surpasses that of cellular DNA.The synthesis of histone mRNA rises concomitantly with the increase in the synthesis of cellular DNA. This occurs in a fashion similar to that observed when confluent CV-1 cultures are stimulated by the addition of fresh serum to the growth medium. However, whereas in cells stimulated with serum the synthesis of histone mRNA closely parallels that of cellular DNA, in cells infected with SV40, histone mRNA synthesis continues at a high rate even after the decline of cellular DNA synthesis. The rate of histone mRNA synthesis thus appears to he coupled to the total (cellular plus viral) DNA synthesis and not to the synthesis of the host DNA alone. The high rate of synthesis of the F1 histone at late times after infection suggests that histone genes are transcribed co-ordinately.     

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.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

Identification of proteins interacting with newly replicated DNA in SV40-infected cells by UV-induced DNA-protein cross-linking

Protein species interacting with newly replicated DNA were analyzed using a photo cross-linking technique. Nascent DNA was labeled in vitro with [alpha-32P]dCTP and BrdUTP in SV40-infected CV-1 cells made permeable with saponin. The labeled cells were then irradiated with UV light (254 nm) and were treated extensively with DNase I. Proteins with radioactive DNA tags were separated by SDS-PAGE and visualized by autoradiography. Among 10-15 proteins which were cross-linked, the proteins with apparent molecular weights of 16.5 K, 44 K, 82 K and those in the 94-140 K region appeared to be associated with newly replicated SV40 DNA. A pulse-chase experiment showed that the 82 K and 94-140 K proteins interacted with new DNA in a relatively localized region close to the replication fork. The 44 K protein was identified as the major viral capsid protein, VP1, using antiserum to SV40 capsid proteins. It was suggested that VP1 binds to nascent DNA shortly after DNA synthesis and migrates into chromatin maturation regions.