Temperature-sensitive Mutant Of SV40 Research Articles

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

Expression Analysis of Several Lymphoma-specific Genes in Human Fibroblasts Immortalized with SV40

Subtraction hybridization was earlier used to obtain cDNA clones corresponding to human genes upregulated in HIV-associated centroblast lymphoma (CL) as compared with HIV-associated immunoblast lymphoma (IL). With inverse subtraction hybridization, clones were isolated that correspond to genes upregulated in IL compared with CL. In addition to cDNAs characterized earlier, the resulting clones contained several (seven CL-specific and three IL-specific) sequences of unknown function. To identify the lymphoma-specific genes that are overexpressed in early carcinogenesis, Northern blotting was used to assess the level of gene transcription in two human fibroblast lines and in their derivatives immortalized with either a temperature-sensitive mutant of SV-40 or with pSV3neo carrying the SV-40 А gene, considering the latter as a model of early cell malignant transformation. Increased expression in at least one immortalized line compared with normal fibroblasts was observed for set, a-myb, ND1, ND2, ND4 (NADH dehydrogenase subunits 1, 2, and 4), COX2, COX3 (cytochrome c oxidase subunits 2 and 3), KIAA0129, and the gene corresponding to hss2-1-7-10 cDNA. High expression of these genes was assumed to be associated not only with lymphomogenesis, but also with early transformation (immortalization) of other, nonlymphoid cells. Expression of the calpain gene and the gene corresponding to hss2-2-9-5 cDNA proved to be lower in immortalized than in normal fibroblasts. This was indicative of an alternative mechanism of fibroblast transformation or of different processes regulating the expression of these genes in early and late carcinogenesis.

The involvement of apoptotic regulators during in vitro decidualization.

The uterus responds to an implanting blastocyst by undergoing extensive tIssue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39 degrees C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33 degrees C. We performed Northern blot analysis for bax, bcl-x(L) and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. We found an increase in the expression of bax when GG-AD cells were grown at 39 degrees C. We also showed apoptosis with Annexin V staining at 39 degrees C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.

The response of adult rat sertoli cells, immortalized by a temperature-sensitive mutant of SV40, to 1,2-dinitrobenzene, 1,3-dinitrobenzene, 2,4-dinitrotoluene, 3,4-dinitrotoluene, and cadmium.

In this study we test the hypothesis that immortalized adult rat Sertoli cells respond to known testicular toxins in a similar manner to Sertoli cells tested in vivo and in primary culture. This cell line was developed by immortalizing adult rat Sertoli cells with the temperature-sensitive mutant of SV40, ts255, such that the cells proliferate at the permissive temperature of 33 degrees C but express differentiated characteristics at the nonpermissive temperature of 40 degrees C. Confluent monolayers, grown at 33 degrees C or 40 degrees C, were exposed to a range of concentrations of dinitrobenzene (DNB) or dinitrotoluene (DNT) isomers or to cadmium chloride. Cellular response was assessed by neutral-red cell viability assay and ultrastructural changes. Cells grown at 40 degrees C were sensitive to lower concentrations of each toxicant than were cells grown at 33 degrees C. 1,2-DNB was more toxic than 1,3-DNB, and 3,4-DNT was more toxic than 2,4-DNT, as judged by the neutral-red cell viability assay. Ultrastructurally, cells treated with 1,2-DNB or 2,4-DNT showed increased numbers of autophagic vesicles compared to controls. Intercellular penetration of ruthenium red demonstrated breached tight junctions in 1,2-DNB and cadmium-treated cells. From these observations, we conclude that this cell line can serve as a model for studying toxic mechanisms in adult Sertoli cells.

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment

Conditional differentiation of heart- and smooth muscle-derived cells transformed by a temperature-sensitive mutant of SV40 T antigen.

To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral infection into rat aortic smooth muscle cells (SMCs) and neonatal heart-derived cells. After G418 selection cell lines isolated were characterized at permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. [3H]Thymidine uptake showed tht progression through the cell cycle is greatly reduced at 39 degrees C. Cytoskeletal proteins, such as actins and vimentin did not change significantly after temperature shift, while the number of desmin-positive SMCs significantly increased when cells were switched to 39 degrees C. Heart-derived muscle cells showed sarcomeric myosin heavy chain reactivity only when grown at 39 degrees C. After thrombin stimulation intracellular calcium in both cell types increased severalfold in 39 degrees C-cells but not in 33 degrees C-cells. Whole cell patch-clamp recordings of SMCs and heart-derived cells revealed a strong increase in nicardipine-sensitive Ca2+ current when cells were switched to 39 degrees C. Nicardipine-insensitive Ca2+ current also increased in both cell types at the non-permissive temperature. Na+ current in SMCs was large at 33 degrees C and small or not detectable at 39 degrees C and absent in heart-derived cells. Using a cDNA probe specific for the alpha 1 subunit of the dihydropyridine-sensitive Ca2+ channel we demonstrate a temperature-sensitive expression of the dihydropyridine receptor mRNA in smooth muscle-derived cells but not in heart-derived H10 cells. Our results suggest that upon downregulation of SV40 T antigen these cells become quiescent and exhibit a more differentiated phenotype. These cell lines may provide a useful tool to investigate ion channel- and receptor signal transduction, as well as cell cycle control in smooth and possibly cardiac muscle cell differentiation.

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes.

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

Expression of gamma-glutamyl transpeptidase in adult rat liver cells after transformation with SV40 virus

By the use of histochemical techniques we have shown that the transformation of rat hepatocytes with simian virus 40 resulted in a large majority of cells producing γ-glutamyl transpeptidase (γGT). Using a temperature-sensitive mutant of this virus we have shown that the precentage of cells exhibiting this enzyme in a temperature-sensitive SV40 mutant is much higher in cells grown at the permissive temperature of 33°C than at the non-permissive temperature of 40.5°C. We thus conclude that, the correlation between γGT expression and chemical or spontaneous transformation of hepatocytes, previously described by other authors, can now be extended to viral transformation and that the enzyme activity is dependent on the expression of a transformed cell phenotype.

Functional interaction between the early viral proteins of simian virus 40 and adenovirus

Complementation studies using early temperature sensitive mutants of SV40 and adenovirus suggest that heterologous DNA-protein interactions can occur between these viruses in coinfected cells. This conclusion is based on the following experimental results: (a) The replication defective adenovirus 5 mutant ts125 can grow in African green monkey kidney cells at the nonpermissive temperature in the presence of preinfection with wild type SV40. (b) The overproduction of early SV40 RNA by the early SV40 temperature sensitive mutant tsA58 can be suppressed by superinfection of the monkey cells with wild type adenovirus. (c) The SV40 helper function for the growth of adenovirus in monkey kidney cells can be provided by the early SV40 mutant tsA58, even at the nonpermissive temperature. We suggest that viral DNA-protein cross-reactions may be responsible for these biological phenomena and show by DNA-DNA hybridization under nonstringent conditions (where hybrid DNAs with up to 35% base mismatch will be thermally stable) that certain regions of the Ad2 and SV40 genomes share significant sequence homology.

The detection and characterization of multiple forms of SV40 nucleoprotein complexes

A procedure has been devised that extracts multiple forms of SV40 nucleoprotein complexes from the nucleus of virus-infected cells. Four distinct complexes have been identified: (i) DNA-replicative intermediates, (ii) a 75S nucleoprotein complex which is the first product of replicative forms, (iii) a previrion structure, and (iv) mature virion. Employing continuous and pulse-chase labeling procedures that follow the incorporation of [ 3H]thymidine into viral nucleoprotein complexes, the kinetics of DNA synthesis and virion maturation are detailed. Replicating SV40 nucleoprotein sediments as a broad complex between 75 and 200 S. The newly synthesized viral DNA is assembled into a 75 S chromosome which appears to mature with time into a 200 S previrion particle. This previrion particle is probably the precursor of the mature virion (250 S). A temperature-sensitive SV40 mutant in the major viral structural protein, VP-1 (tsB-11), fails to produce the 200 S previrion and 250 S virion at the nonpermissive temperature and leads to the accumulation of the 75S nucleoprotein complex. In contrast to previous observations, labeled intracellular SV40 DNA can be efficiently (90%) chased into mature virions. Other published procedures for extracting nucleoprotein complexes ( Green et al., 1971 ; White and Eason, 1971; Varshavsky et al., 1976 ; Christiansen and Griffith, 1977) have not detected this efficient maturation of viral DNA into virions. The reason for this discrepancy is shown to be due to the disruption of mature virions by the buffers, chelating agents, and crude cell extracts employed by these procedures. Disrupted virions produce a heterogeneous set of nucleoprotein complexes that have previously been thought to be natural in vivo complexes.

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.

Transformation of Human Cells by Temperature-Sensitive Mutants of Simian Virus 40

Conditions necessary for the establishment and maintenance of transformation of human cells by wild type and temperature-sensitive mutants of SV40 were examined. For both early and late mutants, the frequency of transformation was found to be up to 5-fold higher, and virus yield 100-fold lower, at 39 degrees than at 33 degrees. No such effect was observed with the wild type virus under the same conditions. This observation is apparently at variance with previously published work, but may be explained by the semipermissive nature of the cells that we used. Increasing the temperature to 40.5 degrees caused cells transformed by the early mutant, tsA30, to lose T-antigen as detectable by staining, and also to lose the ability to grow to high density, while it produced no effect on cells transformed by wild type virus.

Early viral functions of in (ts)-1501 temperature-sensitive SV40 mutant

TEMPERATURE-SENSITIVE (ts) virus mutants have been useful in defining virus–host interactions in SV40 productive and transforming infections. We have isolated an SV40 ts mutant, in(ts)-1501, by extracellular mutagenesis, using ultraviolet radiation of wild type (WT) SV401. The mutant, which has properties different from previously described SV40 ts mutants2,3, is activated from its integrated state in nonpermissive transformed cells at temperatures above 39 °C and is capable of productive infection in permissive cells at elevated (conditional) temperatures1. Many properties of the 1501 mutant are analogous to the early ts cI mutants of bacteriophages in which a heat-inactivated protein cannot maintain the stable integration of the prophage in the bacterial chromosome at elevated temperatures4–8. This analogy suggested that maintenance of SV40 DNA integration in transformed cells requires an early viral function, that is, before the inception of viral DNA replication. Therefore we have investigated the effect of the in gene mutation on two other early virus functions—stimulation of host-cell DNA synthesis and tumour (T)-antigen synthesis.

The temperature-sensitive defect in SV40 group D mutants

Cytoplasmic RNA from cells infected at the restrictive temperature with temperature-sensitive mutants of SV40 has been analyzed by annealing to the separated strands of SV40 DNA. D mutant virions make very little (if any) viral-specific RNA. A mutants make significant amounts of early but not late SV40 RNA. B mutants make both early and late viral RNA. M-band analyses following infection with prelabeled virions suggest that D mutant virions do not form viral DNA-nuclear membrane complexes, whereas those formed by A mutants are normal. Nascent viral DNA is normally released from A mutant complexes at the restrictive temperature.

Biology of SV40 transplantation antigen (TrAg): I. Demonstration of SV40 TrAg on glutaraldehyde-fixed SV40-infected African green monkey kidney cells

The synthesis of papovavirus SV40 transplantation antigen (TrAg) in SV40-infected permissive African green monkey kidney cells (TC7) was demonstrated by using the in vivo transplantation-rejection test. Adult BALB c mice immunized with SV40-infected cells, which had been fixed with 0.25% glutaraldehyde for 5 min at room temperature, rejected a challenge of syngeneic virus-free SV40-transformed mouse cells (VLM). Fixed uninfected TC-7 cells failed to induce transplantation immunity against VLM cells in mice. The induction of SV40 tumor immunity in mice after immunization with glutaraldehyde-fixed infected TC-7 cells was mediated by the TrAg synthesized in permissive monkey cells as a result of virus infection, rather than by the residual infectious virus in the glutaraldehyde-fixed infected cells. The treatment of infectious SV40 with 0.25% glutaraldehyde drastically reduced both viral infectivity and tumor immunity-inducing ability of the virus. Glutaraldehyde-inactivated virus also failed to induce SV40 T antigen in monkey cells, thus ruling out any possibility that the inactivated cell-associated virus was responsible for the induction of tumor immunity in vivo. The amount of infectious SV40 required to induce detectable tumor immunity was found to be 2 × 10 5 PFU of virus administered to adult mice, and is far greater than the amount of virus (1 × 10 4 PFU) which remained associated with SV40-infected monkey cells after fixation of cells with glutaraldehyde. The synthesis of SV40 TrAg in SV40-infected TC-7 cells was inhibited by 5 μg/ml of actinomycin D, but not by 15 μg/ml of cytosine arabinoside, indicating that transcription in the infected cells, but not viral-DNA synthesis, is required for the expression of TrAg. By using 0.25% glutaraldehyde to fix infected cells, it should be possible to test available temperature-sensitive mutants of SV40 for their ability to synthesize TrAg at the nonpermissive temperature, as the glutaraldehyde fixation reduces infectivity of intracellular and extracellular virus, stops the infectious cycle at a particular stage, and preserves immunogenicity of TrAg.

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40 † † Abbreviations used: SV40, simian virus 40; DNA form I, closed circular SV40 DNA; DNA form II, open circular SV40 DNA; endo R ·. Hind, Hpa, Eco, restriction endonucleases from H. influenzae strain d, H. parainfluenzae and Escherichia coli, respectively; dl, deletion mutant; ts, temperature-sensitive mutants. . For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.

Transformation of Primate and Rodent Cells by Temperature-sensitive Mutants of SV40

Cells of mouse, hamster, human and monkey origin were transformed by wild-type SV40 and by temperature-sensitive mutants defective in gene A. The mutant-transformed cells appear to be temperature sensitive for maintenance of transformation. Evidence of reversion of the transformed phenotype of nonpermissive temperatures was obtained in experiments which assayed saturation density, colony formation, and uptake of 2-deoxy-D-glucose. In addition, S antigen modulated in shift experiments while T antigen did not. Based on these results, a model is presented for the mechanism of transformation by SV40.

Characterization of temperature-sensitive mutants of SV40.

SUMMARY We have isolated temperature-sensitive mutants of SV 40 in the lytic system by mutating the virus with nitrous acid or hydroxylamine, and characterized 11 of them. Nine mutants were grouped into two complementation classes in the productive infection of CV-1 cells. The first group contains a single mutant, which was defective in synthesizing virus DNA at the restrictive temperature, but induced the synthesis of cellular DNA. The mutants of the second class produced normal amounts of virus DNA and V-antigen at the restrictive temperature. A naturally-occurring large plaque strain belonged to this group. Both groups retained the transforming ability at the restrictive temperature. Two mutants showed no complementation with either group. One of them had the characters of the first group. The other mutant did not show temperature-sensitivity when the growth was initiated by infection with its DNA. This mutant did not transform 3T3 cells even at the permissive temperature. Rabbit kidney cells, however, were transformed by this mutant effectively at the permissive temperature but less at the restrictive.

Replication of the resident non-defective SV40 in transformed human cells superinfected with DNA from a temperature-sensitive SV40 mutant.

SUMMARY Parental and clonal lines of SV40-transformed human embryonic lung (WI38 Va13A) cells spontaneously produce infectious virus which is temperature-resistant (tr), i.e. replicates in monkey kidney cells at either 38.7 °C or 33.5 °C. Superinfection of WI38 Va13A cells lines with DNA from a temperature-sensitive (ts) SV40 mutant at the permissive temperature (33.5 °C) did not enhance the yield of tr SV40 as would be expected if the superinfecting ts SV40 DNA neutralized an SV40-specific repressor, or if the ts SV40 DNA supplied a virus-specific excision function and/or post-excision functions for the maturation of the resident tr SV40. The failure of superinfecting ts SV40 to enhance replication of the resident tr SV40 of WI38 Va13A cells is not due merely to a competitive advantage of the replicating superinfecting SV40 over the endogenous SV40. Experiments were carried out with 1-β-d-arabinofuranosylcytosine (ara-C) treated and ts SV40 DNA-infected WI38 Va13A cells to permit expression of early ts SV40 functions and to synchronize replication of ts SV40 DNA and the newly excised endogenous SV40 DNA. Ara-C treatment did not enhance formation of the endogenous tr virus. In further competition experiments, CV-1 cells were preinfected with ts SV40 particles and superinfected 6 to 24 h later with tr SV40 DNA recovered from SV40 spontaneously produced by WI38 Va13A cells. Even when the interval between the first and second infection was 24 h, very significant amounts of tr SV40 synthesis occurred and 28% of doubly infected cells still produced temperature-resistant infectious centres. The experiments suggest that rescue of the resident SV40 is determined primarily by cellular rather than superinfecting SV40 gene functions.