SV40-transformed simian cells support the replication of early SV40 mutants

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.     

Temperature sensitive cells in the study of SV40 lysis versus SV40 transformation

Temperature sensitive variant clones of African green monkey kidney cell line (BSC-1) have been isolated which were transformed at a high frequency by SV40 at the restricted temperature, but were lytic to SV40 infection at the permissive temperature. Loss of contact inhibition and cell proliferation at the restricted temperature appeared to be in some way related to the synthesis of T antigen in these variant cell lines.     

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

Introduction of liposome-encapsulated SV40 DNA into cells

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.     

Susceptibility of primate-mouse hybrid cells to SV40

Primate-mouse hybrid cells were challenged with SV40 DNA and monitored for their ability to produce virus. All of the hybrid cells had lost at least half of their primate chromosomes at the time of challenge. Only SV40 T-antigen-positive hybrid cells derived from an SV40-transformed human parental cell produced SV40. This finding suggests that the chromosome(s) necessary for SV40 replication are easily lost on fusion of mouse and primate cells unless the parental cells are already SV40-transformed.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

DNA replication of SV40-infected cells. VII. Formation of SV40 catenated and circular dimers

The binding of methyl isonitrile (CH₃Nandz.tbnd;C) to hemoglobin β chains has been studied by measuring the ¹H nuclear magnetic resonance transverse relaxation times for methyl isonitrile as a function of protein concentration, temperature and ¹⁴N decoupling. Binding of methyl isonitrile both at the heme iron and at a non-specific site (or sites) has an effect upon the measured nuclear spin relaxation times. The results yield a value of 57 ± 12 seconds^?1 (20 °C) for the “off” rate constant K_?1 for specific binding and an Arrhenius activation energy for k_?1 of 14 ± 3 kcal mol^?1.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Purification of SV40 T-antigen by SV40 DNA-sepharose affinity chromatography

T-antigen from SV40-infected BSC-1 cells was purified approximately 30,000 fold using a rapid purification procedure consisting of ammonium sulfate fractionation followed by chromatography on hydroxylapatite, blue-sepharose, and SV40 DNA-sepharose. The SV40 DNA-sepharose was optimized for the binding of T-antigen by the covalent attachment of the SV40 DNA at its BamHI site to cyanogen bromide activated sepharose. The most highly purified T-antigen appeared as a single polypeptide of 94 K daltons by polyacrylamide gel electrophoresis.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

SV40-transformed cells with temperature-dependent serum requirements.

We have isolated temperature-sensitive SV40-transformed 3T3 cells which are unable to grow in low or depleted serum at the nonpermissive temperature. At 39 degrees C, these cells do not grow in 1 percent serum, but they grow if the serum concentration is raised to 10 percent. At 32 degrees they grow in both serum concentrations. This phenotype seems to be due to a cellular mutation, as the virus rescued from these cells is wild-type. We tested whether other characteristics of transformed cells were expressed in a temperature sensitive way. While high saturation density is ts in these cells, other parameters of transformation are expressed at both temperatures. In addition, when these cells are incubated in low serum at 39 degrees C, they keep synthesizing DNA and lose viability very fast, while under the same conditions normal 3T3 cells remain viable for long times and are unable to initiate DNA synthesis. These cells therefore do not appear to revert to a normal phenotype at the high temperature, and they are more likely to represent transformed cell variants with a temperature-dependent serum requirement.