A structural rationale for SV40 Vp1 temperature-sensitive mutants and their complementation

Two groups of temperature-sensitive (ts) mutants, termed ts B and ts C, have mutations in the major capsid protein of SV40, Vp1. These mutants have virion assembly defects at the nonpermissive temperature, but can complement one another when two mutants, one from each group, coinfect a cell. A third group of mutants, termed ts BC, have related phenotypes, but do not complement other mutants. We found that the mutations fall into two structural and functional classes. All ts C and one ts BC mutations map to the region close to the Ca2+ binding sites, and are predicted to disrupt the insertion of the distal part of the C-terminal invading arm (C-arm) into the receiving clamp. They share a severe defect in assembly at the nonpermissive temperature, with few capsid proteins attached to the viral minichromosome. By contrast, all ts B and most ts BC mutations map to a contiguous region including acceptor sites for the proximal part of the C-arm and intrapentamer contacts. These mutants form assembly intermediates that carry substantial capsid proteins on the minichromosome. Thus, accurate virion assembly is prevented by mutations that disrupt interactions between the receiving pentamer and both the proximal and distal parts of the C-arms, with the latter having a greater effect. The distinct spatial localization and assembly defects of the two classes of mutants provide a rationale for their intracistronic complementation and suggest models of capsid assembly.     

Temperature-dependent,neurotrophic factor-elicited,neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33 degrees C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39 degrees C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39 degrees C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33 degrees C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39 degrees C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.     

Novel mast cell lines with enhanced proliferative and degranulative abilities established from temperature-sensitive SV40 large T antigen transgenic mice

Mast cells (MCs) play crucial roles in innate immunity to parasitic and bacterial infections as well as in hypersensitivity, such as the induction and exacerbation of allergy and autoimmune diseases. The regulatory mechanisms for MC development and effector functions are of great interest for developing novel therapeutic strategies against such disorders. Here we report the establishment of novel, immortalized MC lines from bone marrow (BM) cells of a temperature-sensitive mutant of SV40 large T antigen-transgenic mice (termed SVMCs). BM cells from tsSV40LT mice were cultured in the presence of interleukin (IL)-3 for 3 weeks, and then subjected to limiting dilution and single-cell cloning, yielding 27 independent MC clones, three of which were subjected to further analysis. On culture with nerve growth factor, stem cell factor and IL-3, these SVMC clones showed morphologic and biochemical changes from mucosal MC-like to connective-tissue MC-like phenotypes. These SVMC lines exhibited a significantly enhanced proliferation rate, and a higher responsiveness to the high affinity Fc receptor for IgE-mediated intracellular calcium mobilization and degranulation than those of BM-derived cultured MCs. These cell lines should facilitate studies on the mechanisms for the development, differentiation and effector functions of MCs in health and diseases.     

Establishment of SV40-tsA58 transgenic rats as a source of conditionally immortalized cell lines.

To isolate a variety of rat cell lines with differentiated functions, we established transgenic rat lines expressing the temperature-sensitive large T-antigen of simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen itself. We microinjected the DNA into 564 eggs of Wistar rat and 23 independent transgenic candidates were obtained. Ten pups died before weaning and eight transgenic rats could not transmit the transgene to the progeny. Finally, five lines of the transgenic rat were established. Although one line (#1511-6) had low reproductivity, the other four lines reproduced normally. Three out of the four lines (#1507-2, #1509-7, #1519-8) appeared normal but the other line had tumors in the brain and subcutaneous tissue at 3 weeks of age (#1511-6), and in the kidneys and subcutaneous tissue at 18 to 19-weeks of age (#1507-5). Fibroblast cells prepared from transgenic fetuses of lines #1507-5 and #1519-8 expressed the transgene and exhibited temperature-dependent growth. Both of the lines (#1507-5 and #1519-8) were successfully generated to be homozygous by sibling mating of transgenic offspring. These transgenic rat lines have bred through many generations and have been established to be a ready source of novel conditionally immortalized cell lines.     

The differentiation-dependent inhibition of SV40 early promoter/enhancer activity in 3T3-L1 cells is mediated through promoter and not enhancer sequences

Using a plasmid bearing chloramphenicol acetyltransferase (CAT) gene controlled by Simian virus 40 (SV40) early promoter/enhancer complex (pA0cat), we analyzed functional enhancer motifs in 3T3-L1 fibroblast and adipocyte cells. Deletion mutant series of pA0 at the enhancer complex showed that gene expression both in fibroblast and adipocyte cells was dependent on a similar set of enhancer motifs. When pA0 was introduced into 3T3-L1 fibroblasts and the cells were induced to differentiate into adipocytes, CAT activity expressed in fibroblasts was suppressed. Experiments with the deletion mutants at the enhancer complex showed that the suppression was not related to any enhancer motif, and CAT activity was observed with a plasmid having only the promoter sequence. When pA0cat was co-transfected with excess of promoter sequence, the suppression in adipocytes was counteracted. This suggested that negativetrans-acting factors of the promoter sequence were responsible for the suppression in adipocytes.Abbreviations CAT chloramphenicol acetyltransferase - CAT the gene encoding CAT - SV40 Simian virus 40 - Asc-P ascorbic acid phosphate     

Human mutant cell lines with altered RNA polymerase II

A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to α-amanitin at concentrations up to 10 μg/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled α-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50-to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to α-amanitin is due to an alteration of RNA polymerase II.     

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.     

Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.     

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector

Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca⁺⁺-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca⁺⁺-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca⁺⁺-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.     

Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines

Summary A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of -glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH.The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.This work is dedicated to Professor W. Bargmann in honour of his 70th birthday     

Transformation of adult ventricular myocytes with the temperature sensitive A58 (tsA58) mutant of the SV40 large T antigen

Freshly isolated ventricular myocytes have been used extensively as an adult cardiac model system. Due to their inability to undergo cytokinesisin vitro and their dedifferentiated properties in long-term culture, they can not be used for extended studies. Recent reports tell of the establishment of fetal and neonatal cardiac cell lines and the development of adult cardiomyocytes from transgenic animals. A recent report by Kirshenbaum [1], is the first to demonstrate insertion of genes in to adult ventricular myocytes using viral infection. This paper discusses the infection of primary adult differentiated cardiomyocytes with the SV40 large T antigen and subsequent proliferation under temperature sensitive control. Upon further characterization, the cells could be used as a model to study muscle differentiation and repair as well as adult cardiac cell physiology.     

The generation of macrophage-like cell lines by transfection with SV40 origin defective DNA

Two cell lines with properties of mature macrophages have been generated by transfection with SV40 DNA mutated in the origin of replication. One line, BAM, was derived from bone marrow cells from a BALB/c mouse. The other line, BAC1, was derived from splenic adherent cells from a (BALB/c X A.CA) F1 mouse. Both lines produce lysozyme, collagenase, and esterase, bear Fc receptors, and engage in Fc-mediated phagocytosis. Both lines require colony-stimulating factor-1 for continued proliferation. In addition, they express Ia antigens, and may be induced to secrete IL 1. This technique should make possible the generation of Ia-bearing diploid macrophage lines from any strain of mouse. In addition, it may be possible to use this technique to derive monocyte lines from species in which wild-type SV40 DNA causes a lytic infection.     

Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Development and characterization of SV40 immortalized rat parotid acinar cell lines

Summary Rat parotid salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E₁, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.     

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

Summary Rat submandibular salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E₁ were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P_2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.     

Immortalized dendritic cell line with efficient cross-priming ability established from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene

The uptake of an antigen and its presentation to specific T cells by dendritic cells (DCs) is a primary event in initiation of humoral and cellular immune responses as well as the induction of cytotoxic T cells (CTLs). DCs are induced by culturing bone marrow cells in the presence of GM-CSF. However, the resulting DCs are short-lived and the culture usually contains CD11c-negative non-DC cells, which adversely affects reproducibility and makes interpretation of the experimental results difficult. Therefore, it would be useful if DCs could be readily immortalized with their functions being retained. In this study we established a novel, immortalized murine DC line with antigen-presenting capacity in vitro as well as an augmenting effect on humoral and cellular immune responses in vivo, utilizing bone marrow cells from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene. In the presence of GM-CSF, the resulting DC line, termed SVDC, could be continuously subcultured for more than 12 months. When pulsed with OVA alone or OVA-IgG immune complexes via Fcgamma receptors, SVDC augmented OVA-specific T cell proliferation efficiently in vitro, and elicited OVA-specific IgG production in vivo on the adoptive transfer of pulsed SVDC into naive mice. Interestingly, SVDC exhibited significantly high cross-priming ability compared to DCs in a short-term culture, thus leading to their extremely high effectiveness in inducing anti-tumor immunity in vivo. Thus, SVDC is useful for the detailed characterization of antigen presentation, and for research on the various therapeutic benefits of DC vaccination to elicit specific immune responses in immunodeficiencies, infectious diseases and cancer.     

Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer.     

Perturbation of gastric mucosa in mice expressing the temperature-sensitive mutant of SV40 large T antigen. Potential for establishment of an immortalised parietal cell line

Gastric parietal cells have a unique secretory membrane system that undergoes a profound transformation when the parietal cell is stimulated to secrete acid. Understanding this process has been hindered by the lack of an immortalised parietal cell line. Here we have explored a strategy for the development of a parietal cell line by the generation of transgenic mice bearing the temperature-sensitive mutant of the SV40 large T antigen (SV40 tsA58) under the control of the regulatory sequences of the gastric H+/K+ ATPase beta-subunit (H/Kbeta-tsA58). Three H/ Kbeta-tsA58 transgenic mouse lines were established, namely 218, 224 and 228, all of which expressed the tsA58 T antigen in the gastric mucosa. Unexpectedly, the gastric mucosae of all lines were hypertrophic indicating that the temperature-sensitive large T antigen was partially active at 37 degrees C. Immunofluorescence together with light and electron microscopic studies revealed that mature parietal and zymogenic cells were absent in H/Kbeta-tsA58 transgenic lines 218 and 224, and small undifferentiated cells were the dominant cell type in the gastric units. On the other hand, a few mature parietal cells were detected in line 228 together with an increased proportion of undifferentiated cells and, normally rare, pre-parietal cells. As line 228 represented a rich source of pre-parietal cells, gastric cells from line 228 were isolated and cultured at 33 degrees C, the permissive temperature for tsA58. Gastric epithelial cells, expressing the T antigen, were maintained in culture for over 6 weeks. Upon a temperature shift to 39 C the cultured gastric cells developed characteristics of differentiated parietal cells, including the presence of a nascent canaliculus and dramatically increased production of the gastric H+/K+ ATPase beta-subunit. Therefore, this system shows the potential to generate an immature parietal cell line that can be induced to differentiate in vitro.     

Development and characterization of novel cell lines from Etroplus suratensis and their applications in virology, toxicology and gene expression

Four novel cell lines from tissues of eye, gill, kidney and brain of Etroplus suratensis were developed and characterized. The cell lines of eye, gill, kidney and brain were sub-cultured for 245, 185, 170 and 90 passages, respectively, since 2008. These cell lines showed predominantly epithelial-like cells. Effects of temperature and foetal bovine serum concentration on the growth of these cell lines were examined and optimum growth was found at the temperature of 28° C with 20% foetal bovine serum. All the four cell lines were successfully cryopreserved and revived at different passage levels. Cell-cycle analysis of these cell lines was carried out by fluorescence-activated cell sorting. Polymerase chain reaction (PCR) products obtained from the cells and tissues of E. suratensis with primers specific to the conserved region of 16S ribosomal RNA and cytochrome oxidase I genes of E. suratensis revealed the origin of cell lines from E. suratensis. Antibodies raised against the tissues and cells of eye, kidney and gill were highly cross reacted to their specific tissue and cells of E. suratensis. Chromosomal analysis revealed that E. suratensis cells have a normal diploid karyotype with 2n = 48. The cells of these cell lines were successfully transfected with pEGFP vector DNA. The eye (IEE), gill (IEG) and kidney (IEK) cell lines were found to be susceptible to nodavirus but resistant to infectious pancreatic necrosis virus (IPNV). The cells of gill, kidney and eye were applied to test the cytotoxicity of tannery effluents.