Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.     

Comparative study of cytochromes between virus-transformed and untransformed cells

Tissue culture cells of virus-transformed and untransformed cell lines had low contents of cytochromes in the respiratory chain, measured per cell or per mg protein of the cells, in comparison to the cytochrome contents of liver cells in vivo.

In the virus-transformed cells the contents of cytochromes aa₃, b and possibly c₁ were significantly lower than those in the untransformed cells, while the content of cytochrome c was found to be the same or even increased in the transformed cells. Thus, a markedly high ratio of cytochromes c/aa₃ was observed in the transformed cells.

Polarographic measurements of the oxygen uptake have shown a generally low rate of both endogenous respiration and respiration in the presence of glucose and vitamin K₃ in the transformed cells.

The present study indicates that there is a quantitative and possibly qualitative alteration of the respiratory chain components in the transformed cells.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication.

The number of proline residues in a protein should have very marked consequences for the rates of protein unfolding and refolding according to the model proposed by Brandts et al. (1975). Kinetic simulations of this model indicate that the half-time for refolding of a polypeptide chain with 20 proline residues should be greater than 10 minutes and should increase by about an order of magnitude for each additional 10 proline residues. Various means are considered by which the rate of protein folding in vivo and in vitro might be increased.     

Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes.

A method for transferring small DNA fragments from composite polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM)-paper is described. DNA fragments are separated by electrophoresis in polyacrylamide-agarose gels crosslinked with N,N′-diallyltartardiamide instead of N,N′-methylenebis-acrylamide. The crosslinks are cleaved by treating the gel with periodic acid after electrophoresis and the DNA is denatured with alkali. After neutralization, the single stranded DNA fragments are transferred to DBM-paper and detected by hybridization with labeled DNA probes. The procedure has been used to transfer and visualize unlabeled SV40 DNA fragments in the size range 28 to 1790 base pairs.     

Butyrate inhibits mouse fibroblasts at a control point in the G1 phase

Butyrate block 3T6 cells in the G1 phase of the cell cycle approximately 5--6 h prior to the start of the S phase. Serum factors are required before as well as after the butyrate-sensitive steps in G1 in order to allow cells to start DNA synthesis. 3T6 cells infected with SV40 or with polyoma virus are also blocked at the same stage in G1 in the presence of the fatty acid. However, events before as well as after the butyrate-sensitive step do not require serum in virus-infected cells. The sensitivity of the initiation of cellular DNA synthesis to increasing concentrations of butyrate is the same for serum-stimulated or for virus-infected cells. A similar and parallel effect on DNA synthesis is observed if cells are incubated in the presence of very small amounts of cycloheximide. After release of the cycloheximide-induced G1 arrest about 4--6 h have to pass before cells enter the S phase. Cells stably transformed by SV40 are considerably more resistant to low cycloheximide concentrations and to butyrate. These data are discussed in the light of the hypothesis that both low concentrations of cycloheximide and sodium butyrate block cells at a control point in G1 by interference with the synthesis of one or more rapidly turning over, cell cycle-specific proteins.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

Cloning SV40 HindIII restriction fragments into M13 bacteriophage

The six HindIII restriction fragments of the simian virus 40 (SV40) genome were cloned into the bacteriophage M13mp5 vector to generate strand-specific hybridization probes.     

Selection of transfored cells in serum-free media

Summary NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated humanras gene, and the mouse c-myc gene. This work was supported by grants ES-00-210 from the National Institutes of Health (NIEHS) and MV-182 from the American Cancer Society. Dr. L.-C. Chiang is a Visiting Research Scientist from PPG Industries, Inc. Editor’s statement This paper represents a new approach to the identification of oncogenes that would escape the screening methods currently in use. Inherent in the method is the assignment of function to oncogenes.     

Opposite and mutually incompatible structural requirements of type-2 casein kinase and cAMP-dependent protein kinase as visualized with synthetic peptide substrates

Mouse 3T3 cells were grown and synchronized in monolayer with the double thymidine block. Their infection with SV40 took place continuously during the cellular cycle. However, integration of viral DNA into host cell DNA occurred preferentially during the S phase. Phase G1 appeared to be necessary for virus-cell DNA recombination in S phase. Phase G2 did not alter the stability of the integrated viral genome.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

DJ-1, an oncogene and causative gene for familial Parkinson’s disease, is essential for SV40 transformation in mouse fibroblasts through up-regulation of c-Myc

Simian virus 40 (SV40) is a tumor virus and its early gene product large T-antigen (LT) is responsible for the transforming activity of SV40. Parkinson’s disease causative gene DJ-1 is also a ras-dependent oncogene, but the mechanism of its oncogene function is still not known. In this study, we found that there were no transformed foci when fibroblasts from DJ-1-knockout mice were transfected with LT. We also found that DJ-1 directly bound to LT and that the expression level of c-Myc in transformed cells was parallel to that of DJ-1. These findings indicate that DJ-1 is essential for SV40 transformation.

Structured summary

MINT-7988969: DJ-1 (uniprotkb:Q99497) binds (MI:0407) to LT SV40 (uniprotkb:P03070) by pull down (MI:0096) MINT-7988948: LT SV40 (uniprotkb:P03070) physically interacts (MI:0914) with DJ-1 (uniprotkb:Q99LX0) and p53 (uniprotkb:P02340) by anti bait coimmunoprecipitation (MI:0006)     

Identification of cellular components required for SV40 DNA replication in vitro

To investigate the cellular proteins involved in simian virus 40 (SV40) replication, extracts derived from human 293 cells have been fractionated into multiple components. When such fractions are combined with the virus-encoded T antigen (TAg) and SV40 origin containing plasmid DNA, efficient and complete replication is achieved, while each fraction alone is inactive. At present, a minimum of eight such cellular components have been identified. Previous experiments have demonstrated one of these to be the cell-cycle-regulated proliferating-cell nuclear antigen (PCNA). As PCNA has been identified as a processivity factor for DNA polymerase δ, we suggest that both polymerases α and β are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditons, is required with TAg for the formation of a pre-synthesis complex of proteins at the replication origin. The second of these factors, RF-A, is a complex of three polypeptides which may function as a eucaryotic SSB. The third, RF-C, is a factor which is required, with PCNA, for coordinated leading- and lagging-strand synthesis at the replication fork. Complete synthesis and segregation of the daughter molecules also requires the presence of topoisomerases I and II. These results suggest a model for DNA synthesis which involves multiple stages prior to and during replicative DNA synthesis.