Oligomerization of simian virus 40 tumor antigen may be involved in viral DNA replication

Biological implications of the oligomerization of simian virus 40 (SV40) large T antigen for viral DNA replication were studied by using two temperature-sensitive SV40 A-gene mutants, tsA 58 and tsA 1499. Both mutants are defective at elevated temperature for viral DNA replication whereas tsA 58 is like most other tsA mutants additionally heat sensitive for cell transformation. We found that in contrast to tsA 58 encoded T antigen, tsA 1499 T antigen is thermostable in the ability to bind specifically to the origin of replication of SV40 DNA. Detailed structural analysis of tsA 1499 T antigen by sucrose density gradient centrifugation revealed that it is strictly temperature sensitive for the formation of homologous oligomers but, as we reported previously (M. Montenarh, M. Kohler, and R. Henning, 1984, J. Virol, 49, 658-664), not for the association with the cellular phosphoprotein p53. These observations are compatible with the idea that, in addition to the specific origin-binding ability as well as other functional features, the oligomerization of T antigen may be essential for viral DNA replication.     

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

DNA and histone synthesis in butyrate-inhibited BSC-1 cells infected with SV40

The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by 10-fold in the presence of butyrate, and the yield of infectious virus is lowered 100-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones.     

Host cell DNA synthesis as a possible factor in the enhancement of replication of human adenoviruses in simian cells by SV40

Temperature-sensitive mutants of SV40, ts-701 and ts-702, have been isolated after treatment of wild-type virus with nitrous acid and nitrosoguanidine, respectively. Although neither mutant produces infectious virus at the nonpermissive temperature, both can induce SV40 T and V antigens, virus DNA synthesis, and the stimulation of host cell DNA synthesis. Both are late mutants and appear to be in the same SV40 complementation group. Both mutants can also complement the replication of human adenovirus type 7 at the elevated temperature, as can another late mutant, pm-301, and an early mutant, tsA7. Therefore, the adenovirus complementation step appears to be prior to SV40 virus DNA synthesis. Since a BSC-1 clone in which SV40 infection stimulates host cell DNA synthesis allows the complementation of adenovirus type 7 by SV40 while parental BSC-1 cells neither support complementation of adenovirus type 7 by SV40 nor are stimulated to replicate their DNA after SV40 infection, the stimulation of host cell DNA synthesis appears to be critical to adenovirus enhancement. This stimulation appears to be specific since stimulation of DNA synthesis after nutrient deprivation does not induce the replication of human adenoviruses in simian cells.     

DNA replication in synchronized cultured mammalian cells. V. The temporal order of synthesis of component alpha DNA during monkey DNA synthesis induced by SV40 virus.

Purified simian virus 40 (SV40) was used to induce host DNA replication in contact-inhibited monolayer cultures of African green monkey kidney cells. Approximately 20% of the nuclear DNA of these cells is the simple sequence, component α DNA (Maio, 1971). At the beginning of DNA synthesis induced by SV40 viral infection, the average ratio of the specific radioactivity of component α DNA to that of bulk DNA was 0.18. Similar low specific activity ratios are observed early during the DNA synthetic period following the trypsin release of monkey cells from contact inhibition. These results indicate that when host DNA replication is induced by SV40 viral infection, component α DNA synthesis does not shift to precede the replication of bulk DNA.     

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).     

Mapping temperature-sensitive mutants of simian virus 40: Rescue of mutants by fragments of viral DNA

Five temperature-sensitive (ts) mutants of simian virus 40 (SV40), isolated and characterized by Tegtmeyer and Ozer (1971), have been mapped by marker rescue using endo R fragments of wild-type SV40 DNA. For each mutant a specific fragment corrected the ts defect, from which we infer that the mutation is within the genome segment corresponding to the active fragment. Since the position of each fragment in the SV40 cleavage map is known, the mutational sites could be localized. Of the five ts mutants examined, two were “early” mutants and were in complementation group A of Tegtmeyer; these mapped in contiguous Hin fragments H (tsA30) and I (tsA28), which had been shown previously to be part of the “early” region of the SV40 genome. Three ts mutants were “late” mutants and were in complementation group B of Tegtmeyer; these mapped in contiguous Hin fragments F (tsB8), J (tsB4), and G (tsB11), which had been shown previously to be part of the “late” region of the SV40 genome.     

Observations on the resistance of Fpstein-Barr virus DNA synthesis to hydroxyurea

The EBV genome was expressed in EBV “negative” D98/HR-1 somatic cell hybrids following treatment with IUdR as monitored by induction of early antigen (EA), virus capsid antigen (VCA), and virus particles. When the hybrid cells were treated with hydroxyurea (HU) following induction, total DNA synthesis was dramatically reduced but expression of both EA and VCA was not altered. The number of EBV genome equivalents increased following induction with IUdR in the presence of 5 mM HU. Synthesis of EBV specific DNA proceeded to the same degree with or without treatment with HU in lymphoblastoid cells infected with EBV while HU treatment reduced total DNA synthesis by 85–90%. Cytosine arabinoside (ara-C) used in the same manner as HU in IUdR induced hybrid cells dramatically reduced cellular DNA synthesis and totally inhibited EBV DNA synthesis.Of all viruses tested thus far, EBV is unique in that EBV specific DNA synthesis is refractile to inhibition by treatment with HU. Several possible explanations for this unique phenomenon are presented.     

Integration of SV40 DNA and induction of cellular DNA synthesis after a ts SV40 infection

The early temperature-sensitive mutant of simian virus 40 (SV40), ts1101, was characterized in nonpermissive Chinese hamster (ChH) and permissive monkey CV-1 cells for integration of viral DNA and induction of cellular DNA synthesis. At 42° in CV-1 cells, this mutant did not induce the synthesis of T antigen or cellular and viral DNA. The viral DNA did not integrate into the CV-1 DNA, although nuclear penetration occurred. In ChH cells the mutant induced T antigen at 40° and 42°, and the viral DNA integrated into ChH DNA, but ChH DNA synthesis was not induced. These findings suggest that the integration of SV40 DNA does not depend on the replication of cellular DNA and that, at least functionally, integration precedes the induction of cellular DNA synthesis.     

Incorporation of 5-iodo-2'-deoxyuridine (IUdR) into SV40 DNA

The effect of various amounts of 5-iodo-2′-deoxyuridine (IUdR) on Simian Virus 40 (SV40) infected African green monkey kidney (AGMK) cells was investigated in respect to: (1) degree of IUdR incorporation into viral DNA, (2) yield of viral DNA, (3) viral coat protein synthesis, (4) infectivity of IUdR-substituted SV40 DNA.Rather than the absolute concentration it is the ratio of IUdR to thymidine (TdR) in the culture medium which determines the effect of the drug.The degree of IUdR incorporation into viral DNA was linearly dependent upon the relative amount of IUdR in the TdR plus IUdR precursor mixture added to the culture medium. The amount of viral DNA synthesized decreased exponentially with increasing degrees of IUdR substitution. Coat protein formation as determined by immunofluorescence microscopy behaved similar. At equal concentrations of IUdR and TdR, when considerable amounts of viral DNA and capsid proteins were still formed, complete virions were assembled. The infectivity of the IUdR substituted viral DNA was greatly reduced.Illumination studies of the IUdR containing SV40 DNA showed that “visible” light induced single strand breaks into this DNA at neutral pH.The lack of production of infectious SV40 virus in cultures treated with IUdR can be attributed to two major effects: (1) suppression of viral DNA synthesis, (2) reduced infectivity of the IUdR-substituted viral DNA that is produced.     

Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication

Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.     

Regulation mechanism of simian virus 40 late gene expression in primary kidney cells and simian virus 40 transformed 3T3 cells.

Primary and continuous lines of mouse cells are nonpermissive for simian virus 40 (SV40). These cells, infected by the conventional virus absorption method, promote expression of the early but not of the late viral genes. Induction of V-antigen synthesis was found in a large number of primary mouse kidney cells and SV40-transformed $\text{Balb}\text{c}\text{-3T3}$ cells (SV-T2) infected with SV40-DNA component I by the microinjection technique. The proportion of SV40 V-antigen-synthesizing cells is correlated to the quantity of injected DNA molecules in both primary mouse kidney and SV-T2 cells.     

Effect of bovine parvovirus replication on DNA,RNA, and protein synthesis in S phase cells

The kinetics of replication of bovine parvovirus (BPV) and the effect of viral replication on cellular macromolecular synthesis were examined in hydroxyurea (HU)-synchronized fetal bovine spleen cells. Immediately after release of cells from HU block, 80–85% of the cells began to synthesize DNA. The production of infectious progeny BPV proceeded at a faster rate in synchronized cells infected at the beginning of S phase than in asynchronous cultures. In synchronized cells, titers of infectious virus increased at 8 hr p.i. and the maximum titer was achieved by 20 hr. BPV DNA synthesis preceded the production of progeny virus by 2 hr. Although the rates of RNA and protein synthesis in infected cells were severely reduced after 8 hr p.i., BPV replication did not affect the rate of progression of cells through S phase.     

Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences

Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52, 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell.     

DNA replication in SV40-infected cells. IX. The inhibition of a gap-filling step during discontinuous synthesis of SV40 DNA

SV40 DNA apparently replicates by a discontinuous mechanism similar to that proposed for bacteria and bacteriophages. Transient, 4–5 S oligonucleotide intermediates are formed during a brief pulse-labeling with ${}^3$H-thymidine, and these fragments are rapidly joined to preexisting progeny strands (Fareed and Salzman, 1972). An inhibitor of DNA synthesis, hydroxyurea, interferes with the joining of these short 4–5 S oligonucleotide intermediates into longer DNA molecules. This results in the accumulation of replicating forms of SV40 DNA with 4–5 S oligonucleotide fragments hydrogen-bonded at the replication forks to parental DNA strands.Replicating molecules of SV40 DNA isolated from lytically infected cells treated with hydroxyurea were repaired in an in vitro DNA synthesizing system using T4 DNA polymerase and Escherichia coli polynucleotide ligase. The experimental results indicate that at least 80% of the short 4–5 S fragments are separated from themselves or longer progeny molecules by single-stranded gaps in the DNA double helix. These gaps can be repaired in vitro, and the fragments ligased to form longer polynucleotide chains.A model for SV40 DNA replication involving two different DNA polymerases is discussed.     

The SV40 A gene product suppresses the adenovirus H5ts125 defect in DNA replication

The adenovirus H5ts125 mutation is a temperature sensitive defect in the structural gene for a DNA-binding protein required for viral DNA replication. Infection of primary African green monkey kidney cells with SV40, followed by superinfection with H5ts125 at the nonpermissive temperature, permits the replication of adenovirus DNA even in the absence of detectable adenovirus DNA binding protein. The SV40 A gene product is required continuously for the replication of H5ts125 adenovirus DNA. The rate of adenovirus DNA replication in SV40 and H5ts125 doubly infected cells at nonpermissive temperature is about one-half that observed for either H5ts125 singly or SV40-H5ts125 doubly infected cells at the permissive temperature. H5ts125 progeny virus are also produced in the SV40-H5ts125-infected cells at the nonpermissive temperature but the yields are only 3% of those observed in doubly infected cells at the permissive temperature. The suppression of the H5ts125 phenotype by SV40 requires a prior infection of primary or secondary African green monkey kidney cells with a high multiplicity of SV40 (50–100 PFU/cell) and H5ts125 DNA replication is optimally observed between 10 and 15 hr after adenovirus superinfection. These observations suggest that the SV40 A gene product could be involved in the initiation of adenovirus DNA replication thereby bypassing the need for the adenovirus DNA-binding protein.