Wild-type SV40 Research Articles

Human major HSP70 protein complements the localization and functional defects of cytoplasmic mutant SV40 T antigen in Swiss 3T3 mouse fibroblast cells.

The CT3 cytoplasmic localization mutant of SV40 T antigen is neither properly transported to the nucleus nor is it functional in rodent cells. Human precrisis cells are able to complement this mutation, as they are fully transformed by CT3 with wild-type efficiency. The human-specific factors responsible for this species-specific difference in response to CT3 were localized to human chromosome 6 by synteny in a panel of six somatic cell hybrids. A major human HSP70 heat shock protein located on chromosome 6 is expressed constitutively in human cells. Hsp70 proteins have been reported to play a role in intracellular movement of newly synthesized proteins. To test whether human HSP70 played a role in the complementation by human cells of the defect of CT3, we constructed a series of mouse cell lines expressing human HSP70 and tested them for their ability to localize CT3 T antigen in the nucleus and for their ability to be transformed by CT3 DNA. Mouse cell lines expressing human HSP70 protein were able to translocate mutant CT3 T antigen into the nucleus and were transformed by CT3 at rates comparable with wild-type SV40. Mouse-inducible HSP70 protein was not able to translocate cytoplasmic T antigen in Swiss 3T3 mouse fibroblast cells, even after heat shock. Apparently human HSP70 is capable of complementing directly or indirectly the structural and functional alterations in SV40 T antigen introduced by the CT3 mutation.

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.

In vitro translocation through the yeast nuclear envelope: Signal-dependent transport requires ATP and calcium

Abstract Nuclei rapidly purified from yeast Saccharomyces cerevisiae using a cytochalasin B enucleation procedure are substantially free of cell wall, secretory vesicle, plasma membrane vacuolar, and cytoplasmic and mitochondrial contamination. Nuclei obtained in this manner in high yield retain transport properties comparable to nuclei in situ. An in vitro nuclear import assay system has been developed using isolated nuclei and radiolabeled proteins prepared by a coupled in vitro transcription/translation system. Both wild-type SV40 large T-antigen and nucleoplasmin are imported into isolated yeast nuclei, whereas a missense cytoplasmic mutant of the SV40 large T-antigen (Lys128----Thr) and cytoplasmic dihydrofolate reductase are not imported. Association and import of these proteins in a time- and signal-dependent manner resulted in their protection from trypsin that was tethered to agarose beads. Greater than 70% of the labeled protein harboring a karyophilic signal was imported in a reaction that could be blocked by prior treatment of nuclei with trypsin-agarose. Nuclear accumulation of SV40 large T-antigen and nucleoplasmin was unidirectional, ATP and Ca2+ dependent, and was not inhibited by a vast excess of exogenous nonnuclear competitor protein. This system provides an important new tool in combination with powerful yeast genetic methods for analysis of the mechanism and the apparatus for transport at the nuclear envelope.

Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53

SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells.

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones.

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

SV40 DNA in a carrier system of human glioblastoma cells.

The state of the SV40 DNA in a stable carrier system of A172 human glioblastoma cells was examined by Southern blot hybridization analysis. At a sensitivity of 0.1 viral genome equivalents per cell, we detected only free, apparently nondefective, viral genomes. However, when we overexposed our autoradiograms or examined cloned cell populations, integrated viral sequences were observed. Furthermore, aberrant forms of free viral DNA were seen as well. Four clones, isolated at 15 weeks, produced T antigen and displayed enhanced saturation density and plating efficiency characteristic of SV40 transformation. None of these clones produced capsid proteins or infectious virus, even upon fusion with CV-1 cells, Viral DNA in the clones ranged from 0.5 to 50 equivalents per cell, on the average. Two of the Week-15 clones contained a similar (but not identical) predominant truncated SV40 sequence which was present both in a free state and integrated at a single major site in a reiterated head-to-tail array. These clones also contained other minor integrated sequences. Another Week-15 clone contained viral sequences integrated at two major sites as well as heterogeneous free DNA. Only free aberrant DNA was detected in the fourth Week-15 clone. Seven of eight clones isolated at 23 weeks produced no infectious virus or T antigen. No viral DNA was detected in those clones. The eighth clone did produce infectious virus and contained a predominance of free viral DNA. All of the clones were susceptible to superinfection with wild-type SV40, although less so than uninfected A172 cultures.

A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells.

We have constructed a new SV40 mutant, T147, that makes a large T antigen that is only 147 amino acids long. We show that the T147 T antigen is a phosphoprotein that is as stable as wild-type T antigen and that the SV40 viral origin binding activity of the T147 T antigen is reduced at least 100-fold relative to that of wild-type T antigen. Most importantly, we demonstrate that cloned T147 DNA transforms rat F111 cells to anchorage independence as efficiently as cloned wild-type SV40 DNA and that cloned T147 DNA also efficiently transforms C3H10T1/2 mouse cells in a focus assay.

Discrete elements within the SV40 enhancer region display different cell-specific enhancer activities.

The SV40 enhancer contains three genetically defined elements, called A, B and C, that can functionally compensate for one another. By using short, synthetic DNA oligonucleotides, we show that each of these elements can act autonomously as an enhancer when present as multiple tandem copies. Analysis of a progressive series of B element oligomers shows a single element is ineffective as an enhancer and that the activity of two or more elements increases with copy number. Assay in five different cell lines of two separate enhancers containing six tandem copies of either the B or C element shows that these elements possess different cell-specific activities. Parallel oligomer enhancer constructs containing closely spaced double point mutations display no enhancer activity in any of the cell lines tested, indicating that these elements represent single units of enhancer function. These elements contain either a 'core' or 'octamer' consensus sequence but these consensus sequences alone are not sufficient for enhancer activity. The different cell-specific activities of the B and C elements are consistent with functional interactions with different trans-acting factors. We discuss how tandem duplication of such dissimilar elements, as in the wild-type SV40 72-bp repeats, can serve to expand the conditions under which an enhancer can function.

Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes

We have investigated the average nucleosome spacing in the chromatin from several simian virus 40 virion assembly mutants temperature-sensitive in the major capsid protein VP1. Viral assembly intermediates that accumulate in cells infected with mutants that block virion assembly at the propagation step (tsB) have an average nucleosome repeat length similar to that of wild-type SV40 chromatin, approximately 198(± 4) base-pairs. This repeat length is longer than that of the host (BSC-40) cellular chromatin, which has a value of 187(±4) base-pairs. In contrast, SV40 chromatin from cells infected with virus containing a mutation that blocks virion assembly at the initiation step (tsC) has a significantly shorter average repeat length of 177(±4) base-pairs. At the permissive temperature (33 °C), tsC chromatin has a nucleosome spacing periodicity essentially the same as that of wild-type SV40 chromatin. In addition to possessing a chromatin structure with nucleosomes that are, on the average, closer together, tsC chromatin contains a nuclease-hypersensitive or open region in nearly all molecules, but apparently the same number of nucleosomes. These findings suggest that nucleosomes are deposited initially on newly replicated SV40 chromatin in such a way as to leave the DNA region containing the origin of replication and transcription enhancers uncovered. Subsequent interaction with capsid proteins appears to increase the average nucleosome spacing and consequently to cover the open region for encapsidation.

On the functional roles of simian virus 40 large and small T-antigen in the induction of a mitotic host response

The early gene of wild-type (wt) SV40 specifies two related proteins, referred to as large (Mr 88,000) and small (Mr 19,000) T-antigen. Infection with wt SV40 of Go/G1-arrested monkey kidney and CV-1 cell cultures induced in virtually 100% of the cells T-antigen synthesis, followed by a mitotic reaction and the production of SV40 DNA. Parallel cultures were infected with SV40 deletion mutants that produce either no small T-antigen (d1883) or only trace amounts of a truncated form (d1891). Kinetics of synthesis and accumulation of large T-antigen was closely similar to that observed with wtSV40 whereas apparently only 50-60% of the cells participated in the mitotic reaction and the production of viral DNA. These results and those obtained from a comparative study on the abortive (transforming) infection in Go-arrested mouse tissue culture cells indicate that synthesis of large T-antigen alone is sufficient to trigger in 50-60% of the infected cells a mitotic reaction.

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.

The termination region for SV40 DNA replication directs the mode of separation for the two sibling molecules

Separation of the two newly replicated chromosomes in simian virus 40 late replicating intermediates (RI*) occurred by two pathways. The parental DNA strands were completely unwound, releasing circular DNA monomers with a gap in the nascent strand (Form II*), or duplex DNA in the termination region was not unwound, resulting in formation of catenated dimers. Under optimal conditions, both products were transient intermediates in replication, although Form II* was predominant. However, in hypertonic medium both RI* and catenated dimers accumulated, and Form II* was not observed. Hypertonic medium appeared to inhibit both DNA unwinding in the termination region and separation of catenated dimers. When the size of the genome or the position of the origin of replication was changed, termination occurred at sites other than that of wild-type SV40. Neither catenated dimers nor RI* DNA accumulated at these sites. Instead, RI* separated into Form II*. Unwinding parental DNA was more difficult at some termination regions than others. Therefore, although completion of DNA replication does not require a unique termination sequence, this sequence can determine the mode of separation for sibling molecules.

The SV40 T-antigen gene can have two introns

F8dl is an SV40 early-region mutant that lacks over 60% of the DNA sequences normally used to encode large T antigen. This mutant employs a novel splice donor junction at nucleotide 4425 to produce a family of doubly spliced messages. A similar splicing pattern with wild-type SV40 mRNA has been observed, indicating that the wild-type gene for T antigen can also have two introns. A single G-to-T base change at nucleotide 442.5 is sufficient to eliminate this novel donor splice junction.

SV40 vector with early gene replacement efficient in transducing exogenous DNA into mammalian cells.

An early replacement SV40 vector, SV40-Mu beta, was constructed by replacing part of the early genes of the virus with mouse interferon-beta (IFN-beta) cDNA. Upon transfection of COS-7 cells with this DNA, transducing viral particles were produced, which could infect various cells and cause efficient production of mouse IFN-beta. The viral stock contained no detectable wild-type SV40. The IFN production after the virus infection was very high in monkey kidney cells, but less so in human epithelial cells, and low in mouse, pig, hamster cells and in human lymphocytes. The efficiency of introduction of the DNA to monkey kidney cells was compared with that by the calcium phosphate precipitation method, and the viral vector was found to be more efficient by a factor of several tens to hundreds.

Cloning of SV40sv40 genomes from human brain tumors

From two human brain tumors SV40 genomes were isolated by recombinant DNA techniques. The SV40 genome cloned from a human meningioma DNA was shown to be indistinguishable from wild-type SV40. In contrast, the SV40 genome cloned from a human astrocytoma proved to be a nonviable deletion mutant with a truncated early region removing most of the large T-coding region. In addition, this mutant also carries a tandem duplication of an intact origin of replication.

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.

Structural comparisons of wild-type and nuclear transport-defective simian virus 40 large tumor antigens

PARA(nT) is a defective SV40-adenovirus 7 hybrid virus which contains the entire early region of the SV40 genome and codes for the synthesis of SV40 large tumor antigen (T-ag). A transport-defective variant of this hybrid, PARA(cT), encodes T-ag that is not transported to the nucleus, but accumulates in the cytoplasm. The structures of T-ags extracted from wild-type (WT) SV40-, PARA(nT)-, and PARA(cT)-infected cells were compared by peptide mapping. All three types of T-ag underwent considerable degradation when extracted using Tris-buffered Nonidet P-40 at pH 8.0. The addition of 200 μM leupeptin to the extraction buffer significantly inhibited this degradation. Comparison of methionine-containing tryptic peptides revealed no differences among the T-ags, suggesting that their primary structures are similar or identical. Phosphopeptide mapping revealed no differences between SV40- and PARA(nT)-encoded T-ags. In contrast, PARA(cT)-encoded T-ag lacked a prominent phosphopeptide that was present in both of the others. The possible relevance of this difference in phosphorylation to the transport defect is discussed.

SV40 immortalization of adult human mesenchymal cells from neuroretina. Biological, functional and molecular characterization.

Human adult mesenchymal cells from neuroretina (human choroid cells, HC) have acquired an infinite lifespan, following phenotypic transformation with a wild-type SV40. Immortalized cells (HC/SV40) contain high numbers of free circular viral DNA, and integrated molecules in a head-to-tail array in the cellular DNA. HC/SV40 cells express both the virus-coded "T" antigens and the cell-coded p53 transformation-associated protein. The transformed phenotype was further characterized by loss of contact inhibition of cell division, inability to induce the retraction of a fibrin clot and to spread within fibrin, and the existence of an altered distribution of actin cables. For the first time we also describe a coupling of the immunofluorescence and the quantitative cytofluorometric analyses, a new transformation parameter, since we show that SV40 transformation causes reorganization of the cell membrane by inducing the unmasking of the antigen recognized by the 4F2 monoclonal antibody, which is present in a "cryptic" form in the untransformed cells. Though the HC/SV40 cells have been continuously passaged over a 3-year period, they have not yet achieved a fully malignant phenotype, since they retain serum-dependency and the presence of a well developed fibronectin pericellular network, and they are not tumorigenic in nude mice. Thus this human immortal cell line constitutes a very useful tool for studying the progression toward full malignancy and the relationships between evolution of transformation parameters and changes in the viral and cellular genome interplay.

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.

Transformation of mouse mammary epithelial cells by papovavirus SV40

Primary and established murine mammary epithelial cells and wild-type SV40 were employed to study the phenomenon of epithelial cell transformation. Thirteen independent transformed cell lines were derived. All contained SV40 intranuclear T antigen. Eight transformed mammary cell lines were examined ultrastructurally and all were found to exhibit pronounced epithelial cell characteristics, including desmosomes and tight junctions. Growth studies revealed that while normal mammary cells were unable to grow in low serum (2% FBS), established Cl S1 mammary cells and SV40-transformed mammary epithelial cells replicated well. Cell densities achieved by the transformants were only slightly elevated in high serum (13% FBS) over normal cell values. All the transformants formed colonies on plastic and exhibited anchorage-independent growth in methylcellulose. Five of the transformed lines were tumorigenic in syngeneic animals, in marked contrast to the lack of transplantability usually observed with SV40-transformed mouse fibroblasts. Anchorage-independent growth was not a predictor of tumorigenic potential in this system. The transformants exhibited a spectrum of responsiveness to exogenous growth factors. This study establishes that the SV40-murine mammary cell system is a valid model for analyses of the process and consequences of epithelial cell transformation, in general, and mammary cell transformation in particular.