Persistence and recombination of minicircular SV 40 DNA in permissive cells

Summary Minicircular SV 40 DNA molecules were obtained by serial undiluted passages of the virus stock. By using high multiplicities of infection, and by cocultivation of infected and uninfected cells, as much as 90% of the SV 40 DNA molecules were deleted. The deletions were confined, as shown by restriction enzyme analysis, preferentially to the late region of the genome. Three size classes of minicircular SV 40 DNA molecules were separated electrophoretically (mini-1, 10–15% deleted; mini-2, 10–20% deleted, and mini-3 20–25% deleted) and used for transfection of permissive CV-1 cells. The cell cultures were examined after various periods of time for the expression of SV 40 tumor and capsid antigens, the replication of viral DNA, the presence of infectious virions and for the rescuability of minicircular DNA after super-infection with wild-type helper SV 40-virus. When helper virus was used to coinfect CV-1 cells together with either mini-1,-2, or-3 DNA, all classes of minicircular DNA were capable of replicating. This complementation effect was abolished, however, in the case of mini-2 and-3-infected CV-1 cells, when the superinfection with helper virus was performed one to two weeks after the initial transfection with minicircular DNA. In the absence of helper virus, only mini-1 DNA could replicate and give rise to infectious virus progeny.Cells which were infected with either mini-2 or-3 DNA displayed no phenotypic alteration for up to one month. Immunofluorescence tests for SV 40 tumor and capsid antigens were negative, and we also could not detect the presence of ³H-labeled superhelical SV 40 DNA. Between 5 and 8 weeks after infection, however, a cytopathic effect developed, and SV 40-gene functions such as virus-specific antigens, replication of SV 40 DNA and appearance of viral progeny were expressed. The size of the newly generated intracellular superhelical SV 40 DNA corresponded predominantly to the size of the minicircular DNA class which had been used for transfection (either mini-2 or mini-3), but larger molecules also appeared (up to the size of wild-type FO I DNA). The restriction enzyme cleavage patterns differed both from the profiles of the wild-type DNA and from mini-2 and mini-3 DNA, respectively; the main difference being that a population of SV 40 genomes had evolved in which sequences from the late region, which were underrepresented in the mini-2 and-3 DNA, had accumulated. This shows that viral DNA molecules containing deletions, which have persisted in the cell for a prolonged period of time in an unexpressed state, can subsequently recombine to form DNA molecules which can give rise to infectious virions.     

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.     

Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.     

Simian virus 40-related antigens in three human meningiomas with defined chromosome loss.

Two out of seven meningiomas tested in early cell cultures by indirect immunofluorescence staining showed simian virus 40 (SV40)-related tumor (T) antigen. In one tumor 90% of the cells were positive. An additional SV40-related antigen (U) was found in 10% of cells of a third tumor. These findings indicate that the meningioma cells showing a positive reaction are transformed by a papova virus that has at least partly the same antigenic properties as SV40 virus. SV40-related viral capsid (V) antigen was absent in all the meningiomas tested. No virus infectious for African green monkey kidney (AGMK) cells could be isolated. The tumors positive for T and U antigens showed the chromosome aberration typical for human meningiomas, i.e., the loss of one chromosome, G-22. The T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive meningioma cells were performed: fusion of cells pretreated with 8-azaguanine with cells premissive for SV40 led to a low percentage (0.01-0.05%) of V-antigen-positive nuclei in heterokaryon cultures. On the basis of these results, the possibility of a correlation between the meningioma, a relatively common intracranial tumor in man, and an SV40-related papova virus must be considered. It remains to be shown whether this virus is a causative agent for human meningiomas.     

The Isolation of Simian Virus 40 Variants with Specifically Altered Genomes

Serial passage of simian virus 40 (SV40) at high multiplicity of infection leads to the emergence of variants with deleted, substituted, and/or duplicated DNA. Individual variants have been cloned by selective complementation with temperature sensitive SV40 mutants, or nonselectively by coinfection of cells with wild-type helper virus. In each case, the presence of variants was detected by the appearance of discrete short viral genomes in infected cell lysates. Such short genomes, isolated by agarose gel electrophoresis, were shown to be specifically altered by comparing the electrophoretic pattern of their DNA fragments produced by Haemophilus influenzae restriction endonuclease with the pattern of fragments from parental DNA. In addition to defective variants, one infectious variant that had an additional segment of DNA within its genome was isolated.     

Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells

Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple genetically marked viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene.In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.     

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.     

Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells.

Exposure of simian virus 40 (SV40)-transformed Chinese hamster embryo cells to various chemical and physical carcinogens induced SV40 DNA synthesis. Although the carcinogen-mediated amplification of SV40 DNA is regulated by the viral A gene, the induction of viral DNA synthesis does not result in the rescue of infectious virus or the formation of complete viral DNA molecules. Instead, a heterogeneous collection of DNA molecules containing SV40 sequences was generated by treatment with 7,12-dimethylbenz[a]anthracene. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant showed that not all sequences in the integrated SV40 inserts are present. The possibility that amplification of SV40 sequences is a reflection of a general-gene-amplification phenomenon mediated by carcinogens is discussed.     

Detection of SV40 like viral DNA and viral antigens in malignant pleural mesothelioma.

This study investigated the presence of simian vacuolating virus 40 (SV40) deoxyribonucleic acid (DNA) in malignant pleural mesothelioma, non-neoplastic mesothelium and pleural carcinoma metastasis and correlated these data with immunohistochemistry for SV40 viral antigens. The novel Primed In Situ (PRINS) method was applied to detect the presence of SV40 DNA in situ in tissue sections of malignant mesothelioma (n = 25), non-neoplastic mesothelium (n = 30) and pleural carcinoma metastasis (n = 30). Immunohistochemistry with an SV40-specific antibody was applied for detection of the SV40 viral antigen in the same material. SV40 DNA and expression of one of the viral proteins (small t-antigen) was found in approximately 60% of the investigated mesothelioma cases in contrast to non-neoplastic mesothelium and carcinoma metastasis that were negative for both SV40 DNA and SV40 viral antigens. These results suggest that simian vacuolating virus 40 deoxyribonucleic acid may be biologically active as there was also immunoreactivity for simian vacuolating virus 40 viral antigen in those cases positive for simian vacuolating virus 40 deoxyribonucleic acid with the primed in situ reaction. Simian vacuolating virus 40 viral deoxyribonucleic acid and antigens may be potential markers for neoplastic mesothelium that may prove useful in the rather difficult histopathological differential diagnosis between malignant mesothelioma and reactive mesothelium or pleural carcinoma.     

Simian virus 40 T antigen is required for viral excision from chromosomes.

We describe experiments that show that simian virus 40 (SV40) T antigen is required for viral excision from host chromosomes at some point prior to or during the homologous recombination events that create circular wild-type virus. Two recombinant SV40-pBR322 plasmids were constructed such that homologous recombination across similar-sized but different duplications of SV40 would reconstitute wild-type viral DNA. One plasmid (pSVED) was constructed such that the duplication separates the viral early T-antigen promoter from the coding sequences; the other recombinant (pSVLD) contains a duplication of the late viral sequences and thus maintains a complete T-antigen gene. These plasmids were individually established in Rat 2 cells via cotransformation with the herpes virus Tk gene. Both classes of cell lines contained integrated tandem arrays of the plasmids and yielded equivalent levels of infectious virus after cell fusions with COS-7 cells; however, only the T+ lines yielded virus after cell fusion with CV-1 cells. These results are consistent with the notion that viral excision is initiated by T-antigen-mediated in situ replication of viral DNA as proposed in the "onion skin" model. In contrast, both plasmids yielded infectious virus when transiently introduced via transfection into CV-1 cells. This latter finding is discussed in terms of the possible induction of cellular repair and recombination pathways evoked by the introduction of damaged DNA into the nucleus.     

Viable deletion mutants of simian virus 40: selective isolation by means of a restriction endonuclease from Hemophilus parainfluenzae

Resistance of simian virus 40 (SV40) DNA to cleavage by Hemophilus parainfluenzae II (HpaII) restriction endonuclease has been used as a positive, in vitro selection for mutants lacking the one HpaII endonuclease-cleavage site of wild-type SV40 DNA. Each of 10 viable mutants isolated by this procedure multiplies significantly more slowly than wild-type virus and contains a small deletion (80 to 190 base pairs in size) of the region of the genome that includes the HpaII endonuclease-recognition sequence. These well-defined mutants, having a selective disadvantage for growth, would not have been readily obtained by conventional methods used to screen for viral mutants. Therefore, in certain circumstances, restriction endonucleases are effective reagents for the selection of new classes of mutants. Because these small deletions can be visualized in heteroduplexes, these mutants provide internal markers for mapping other alterations or features of the simian virus 40 genome.     

Simian virus 40 efficiently infects human T lymphocytes and extends their lifespan

The relevance of viral infections to the onset and progression of human hematologic malignancies and other blood diseases is still a matter of active investigation. Purified human T lymphocytes isolated from the peripheral blood mononuclear cells of healthy blood donors were experimentally infected with simian virus 40 (SV40), a small DNA tumor virus. SV40-positive T lymphocytes extended their lifespan up to day 80 postinfection (PI). Expression of viral antigens, such as the large T antigen and the viral capsid protein VP1 from the early and late regions, respectively, was detected up to day 40 PI. SV40 viral progeny were continuously produced from day 10 to 40 PI. SV40 DNA sequences were detected in infected T cells for up to 80 days. Our data indicate that human T lymphocytes can be efficiently infected with SV40. Although T cells infected by SV40 were not immortalized, 30% of these lymphocytes appeared to be morphologically transformed with an enlarged T-cell shape. Our investigation provides a simple model for studying the interactions of human T lymphocytes with this small DNA tumor virus and it might represent an experimental tool for investigating new biomarkers and targets for innovative therapeutic approaches.     

Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA.

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.     

Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.

Rat-1 cells were transfected with the restriction enzyme fragment of polyoma virus DNA that extends clockwise from the Bcl I site ((65.4 map units) to the EcoRI site (0/100 map units). Six transformed cell lines were obtained and one of them (BE-1) has been investigated in detail. The viral DNA that is integrated into host DNA in this line appeared to consist of two fragments arranged in a "head-to-tail" tandem with no detectable intervening host sequences. BE-1 cells contained polyoma virus small and middle tumor antigens that were indistinguishable from the corresponding tumor antigens from lytically infected cells. No large tumor antigen was detected but a "new" Mr 34,000 protein, which proved to be a truncated version of large tumor antigen, was immunoprecipitated by anti-tumor-antigen antiserum. After injection of 10(6) BE-1 cells into young syngeneic Fischer rats, tumors appeared within 3--4 weeks. Thus, the coding capacity of the Bcl I/EcoRI fragment of polyoma virus DNA is sufficient to enable the cells to produce all of small and middle tumor antigens and about a third of large tumor antigen, to transform cells stably in culture, and to produce tumors in vivo.     

Location of histones on simian virus 40 DNA.

The physical location of histone molecules in a simian virus 40 DNA-histone complex isolated from purified virions was examined using site-specific restriction endonucleases. The complex contains four host histone species but lacks histone F1. Histones prevent complete cleavage of SV40 DNA by two restriction enzymes, HindIII and EcoRI. From the pattern of DNA fragments resulting from cleavage of the histone-DNA complex by the HindIII endonuclease, which makes six breaks on purified SV 40 DNA, we have concluded that histones are randomly arranged on SV40 DNA relative to restriction enzyme cleavage sites. The EcoRI endonuclease, which makes one break in SV40 NDA, was used to determine the degree of physical coverage of the SV 40 DNA molecule by histones. We observed that 80% of the EcoRI sites in the complex are accessible to the enzyme while 20% are "closed." This degree of coverage is consistent with the mass ratio of DNA:histone in the complex as revealed by the buoyant density of the formaldehyde-fixed complex. We conclude that the histones in the complex are located randomly on the SV 40 genome and cover approximatley 20% of the DNA. These results suggest that the histone species F2b, F2al, F2a2, and F3 are bound without regard to nucleotide sequence of SV 40 DNA.     

Instability of integrated viral DNA in mouse cells transformed by simian virus 40

The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.     

Cytoplasmic transfer of DNA containing simian virus 40 sequences into mouse 3T3 cells.

This paper describes the rare cytoplasmic transmission of defective simian virus 40 (SV40) viral DNA from enucleated cells (i.e., cytoplasts) of the SV40-transformed mouse cell line SVT2 (chloramphenicol-resistant) into cybrid cells formed by fusion of these cytoplasts with BALB/c 3T3 cells (thymidine kinase-deficient). The cybrids were selected in medium containing 1% serum, bromodeoxyuridine, and chloramphenicol. They were identified by their 3T3 chromosome content, by the instability of tumor (T)-antigen expression, by their transformed phenotype, and by their drug resistance. The yield of rare cybrids was about 5 x 10(-7) 0.1% of the yield on medium with 10% serum. The presence of the SV40 genome was detected by the expression of SV40-specific T antigen and confirmed (unpublished data) by hybridization of viral DNA probes with restriction enzyme fragments of nuclear DNAs from cybrid clones. Restriction site mapping (unpublished data) showed that at least 1 kilobase of host flanking DNA on each side of the SV40 DNA was included in the transferred segment. The transforming DNA was not stably integrated initially, as judged by cellular heterogeneity in T-antigen expression. Stable T-antigen-positive and negative subclones were recovered in 10% serum; instability could be retained for at least 30 doublings during growth in 1% serum. The instability is interpreted as evidence of non-integration or unstable integration of the transferred DNA into the host genome. The cytoplasmic transfer is interpreted as evidence that chromosomal fragments or intact chromosomes can be transferred rarely through the cytoplasm in cybrid crosses.