Analysis of nonpermissivity in mouse cells overexpressing simian virus 40 T antigen.

To analyze the nature of the nonpermissivity of mouse cells for simian virus 40 (SV40) DNA replication, we isolated mouse cells producing SV40 T antigen (Tag) at levels equal to or greater than that found in COS1 cells. These mouse cells were nonpermissive for the replication of exogenously added SV40 DNA, although purified Tag isolated from these cells was able to support SV40 DNA replication in vitro. Furthermore, when mouse cells expressing Tag were fused to monkey cells, SV40 DNA replication was observed. These results indicate that the mere production of large quantities of wild-type SV40 Tag does not overcome the block of nonpermissivity in mouse cells and that cellular factors must play a critical role.     

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.     

The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen.

Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re-enter the cell cycle and progress through G1 into the S phase. Genetic analysis of T antigen indicated that neither its Rb/p107-binding activity nor its p53-binding activity is essential to induce DNA synthesis in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells into the S phase than did wild-type T antigen. Inactivation of both activities resulted in a T antigen essentially unable to induce DNA synthesis. Missense mutations in either the DNA-binding region of the N terminus also impaired the ability of full-length T antigen to stimulate DNA synthesis in CV1P cells. The wild-type kinetics of cell cycle progression were restored by genetic complementation after coinjection of plasmid DNAs encoding different mutant T antigens or coinjection of purified mutant T-antigen proteins, suggesting that the four mitogenic functions of T antigen are independent. The maximal rate of induction of DNA synthesis in secondary primate cells and established rodent cell lines required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycle progression is presented.     

A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G₁ phase cells and increases the percentages of S and G₂ + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.     

Quantitative correlation between simian virus 40 T-antigen synthesis and late viral gene expression in permissive and nonpermissive cells

The time-course of intranuclear Simian virus 40 (SV40) tumor (T) antigen synthesis and accumulation in permissive CV1 monkey cells and nonpermissive 3T3 mouse cells has been studied by immunofluorescence and cytofluorometry. CV1 cells accumulate T antigen continuously over a period of 48 h after infection, whereas in 3T3 cells the T-antigen content remains about constant and at a comparatively low level. Only those CV1 cells which have attained a threshold concentration of intranuclear T antigen synthesize viral capsid proteins (V antigen). In nonpermissive 3T3 cells, the T-antigen threshold value for the onset of V-antigen synthesis is higher than in CV1 cells and is never reached by infected cells. However, 3T3 cells microinjected with sufficient amounts of SV40 DNA easily surpass this value and behave permissively.     

Reintroduction of a normal retinoblastoma gene into retinoblastoma and osteosarcoma cells inhibits the replication associated function of SV40 large T antigen

The product of the retinoblastoma (Rb) gene can form complexes with the transforming proteins of small DNA tumor viruses, including SV40 large T antigen (Tag), adenovirus E1A, and the human papilloma virus E7. The strong correlation between their ability to transform and their ability to bind Rb protein suggests that these oncoproteins exert their effect through blocking the Rb function. SV40 Tag causes oncogenic cell transformation of rodent cells, and it is also required for viral DNA replication. In this paper, we investigated the effect of the Rb protein on the SV40 replication associated function of Tag. We present evidence suggesting that the complex formation between Rb and Tag interferes with the viral DNA replication. In Y79 retinoblastoma and Saos-2 osteosarcoma cells, which lack functional Rb protein, a SV40 based plasmid vector, pSVEpR4, replicates well. In the same cells reconstituted for Rb expression with an intact Rb gene introduced by retroviral mediated gene transfer, pSVEpR4 replicates to a considerably lower level. The inhibitory effect of Rb protein was surmounted by increasing the intracellular level of Tag. Increasing amounts of Tag in wild-type Rb negative Y79 cells had virtually no effect on SV40 replication. Furthermore, the overexpression of Tag in Rb reconstituted Y79 cells did not alter the growth rate of the cells. These data suggest that Rb protein interacts with Tag and modulates its ability to promote SV40 DNA replication.     

CD4+ T lymphocytes are critical mediators of tumor immunity to simian virus 40 large tumor antigen induced by vaccination with plasmid DNA

A mechanistic analysis of tumor immunity directed toward the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory for scenarios of recombinant Tag immunization in BALB/c mice. In the present study, we performed a preliminary characterization of the immune components necessary for systemic tumor immunity induced upon immunization with plasmid DNA encoding SV40 Tag as a transgene (pCMV-Tag). Antibody responses to SV40 Tag were observed via indirect enzyme-linked immunosorbent assay following three intramuscular (i.m.) injections of pCMV-Tag and were typified by a mixed Th1/Th2 response. Complete tumor immunity within a murine model of pulmonary metastasis was achieved upon two i.m. injections of pCMV-Tag, as assessed by examination of tumor foci in mouse lungs, without a detectable antibody response to SV40 Tag. Induction-phase and effector-phase depletions of T cell subsets were performed in vivo via administration of depleting rat monoclonal antibodies, and these experiments demonstrated that CD4(+) T lymphocytes are required in both phases of the adaptive immune response. Conversely, depletion of CD8(+) T lymphocytes did not impair tumor immunity in either immune phase and resulted in the premature production of antibodies to SV40 Tag. Our findings are unique in that a dominant role could be ascribed to CD4(+) T lymphocytes within a model of DNA vaccine-induced tumor immunity to Tag-expressing tumor cells. Additionally, our findings provide insight into the general mechanisms of vaccine-induced tumor immunity directed toward tumors bearing distinct tumor-associated antigens.     

Molecular quantification of cell cycle-related gene expression at the protein level

BACKGROUND: Immunofluorescence cytometry of antigen and DNA content provides relative measurements of the cell cycle phase distribution of a specific epitope. Measurement of correlated expression of epitopes on signaling and regulatory proteins will be useful in the study of the complex pathways involved in cell cycle regulation and carcinogenesis. However, to formulate regulatory pathway models, measurements of molecules per cell would be more useful than relative measurements of intensity. Here, we report on a system in which the relationship between molecules and fluorescence is determined for a reference set of cell lines that are then used to directly calculate the number of molecules for unknowns. To demonstrate the process, we calculated the cell cycle phase distribution of SV40 large T antigen (Tag) in the reference cells. METHODS: A set of cell line clones expressing different levels of Tag were isolated. Quantitative Western blots of these cells and purified, recombinant Tag were performed. Cells from the same sample were stained and analyzed by flow cytometry for Tag and DNA. The relationship between molecules and fluorescence was established and calculations were performed for the phase distributions of Tag. RESULTS: The five cell lines had 0.11, 0.27, 1.06, 2.44, and 2.63 x 10(6) molecules of Tag per cell, determined by Western blot. The average coefficient of variation was 10.6%. The relationship of molecules to fluorescence fit a linear equation (r(2) = 0.96) over the range, 0.11 - 2.63 x 10(6) molecules, however, the same equation did not fit the relationship between 0 molecules, defined by isotype staining controls, and the lowest expressing cell line. To calculate the phase distributions of molecules in the lowest cell line, a second linear equation from 0 to 110,000 molecules was used. CONCLUSIONS: This work describes a system where fixed cells expressing various levels of a target antigen quantified by Western blots can be used to standardize flow cytometric measurements of gene expression in absolute terms.     

Differential ability of fixed antigen-presenting cells to stimulate nominal antigen-reactive and alloreactive T4 lymphocytes

The capacity of paraformaldehyde-fixed human antigen-presenting cells (APC) to induce responses by autologous, freshly isolated peripheral blood T4 cells was examined and was compared with their ability to stimulate allogeneic T4 cell DNA synthesis. Fixation of glass-adherent cells (AC) with as little as 0.06% paraformaldehyde abolished leucine incorporation, whereas fixation with 0.75% paraformaldehyde caused death of greater than 98% of the AC. Control APC were able to take up and present the soluble antigens streptokinase-streptodornase (SK-SD), tetanus toxoid, or tuberculin-purified protein derivative to autologous Ia-depleted T4 cells. Fixation with greater than 0.06% paraformaldehyde eliminated such ability. When AC were incubated with antigen overnight and were then fixed, however, they were able to present nominal antigen to autologous T4 cells in a genetically restricted manner that was blocked by monoclonal antibodies directed against monomorphic determinants on class II major histocompatibility complex (MHC) molecules. Despite the ability to present nominal antigen, paraformaldehyde-fixed AC were unable to induce allogeneic T4 cell proliferation. Similar results were observed when non-T cells or spleen cells were used as stimulators. The inability of fixed APC to stimulate allogeneic T4 cell DNA synthesis was not reversed by increasing the number of fixed APC or by the addition of control AC autologous to the responding cells. Moreover, interleukins 1 and 2 either alone or in combination also failed to permit maximal T cell proliferation in response to fixed allogeneic APC. The differential effects of fixation on nominal antigen and alloantigen presentation could not be explained by the loss of membrane thymocyte stimulatory activity on fixed AC. These results indicate that antigen-bearing fixed APC are competent to stimulate proliferation by antigen-reactive T4 cells, but are deficient at inducing allogeneic T4 cell DNA synthesis. The differential sensitivity of these two Ia-restricted functions of APC to chemical denaturation (reductive methylation) by paraformaldehyde suggests that the allodeterminants and restriction elements for nominal antigen on MHC class II molecules can be functionally dissociated.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Study of the Functional Activities Concomitantly Retained by the 115,000 Mr Super T Antigen, an Evolutionary Variant of Simian Virus 40 Large T Antigen Expressed in Transformed Rat Cells

Simian virus 40 (SV40) transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) do not synthesize normal-size large T antigen (M(r), 90,000); instead, they produce a 115,000 M(r) super T antigen (115K super T antigen). This super T antigen is SV40 virus coded, and its synthesis results from rearrangement and amplification of integrated viral DNA sequences in subclone 7 (May et al., Nucleic Acids Res. 9:4111-4128, 1981). In this study the functional activities of 115K super T antigen were compared with the functional activities of SV40 large T antigen. Transfection experiments were performed with (i) cosmid SVE 5 Kb and plasmid pSVsT, both containing the super T antigen gene and (ii) plasmids pSV1 and pSV40, both containing the large T antigen gene. Transfection of pSVsT DNA or SVE 5 Kb DNA into secondary cultures of rat kidney cells induced the formation of transformed cell foci with an efficiency that was about 50% of the efficiency of pSV1 DNA or pSV40 DNA. Concomitant with the transforming activity, two other activities were also retained by super T antigen, namely, the ability to enhance the level of host cellular protein p53 and the capacity to bind to p53. In contrast, pSVsT and SVE 5 Kb DNAs were markedly deficient in the capacity to support tsA58 DNA replication in CV1-P cells at a nonpermissive temperature (41 degrees C), as shown by cotransfection experiments. The yield of virus produced in these experiments was 400-fold less than the yield obtained in parallel experiments with pSV40 or pSV1. However, SVE 5 Kb and pSVsT have a functional SV40 replication origin, as shown by their efficient replication in COS 1 cells which provided functional large T antigen. Super T antigen also possesses a specific affinity for sequences of SV40 viral origin. Our results suggest that under certain conditions, evolutionary changes in T antigen take place and that these changes could be restricted to the phenotypic requirement of maintaining a structure that is able to induce cell transformation, to form a complex with p53, and to enhance the cellular level of p53. Therefore, there appears to be a close relationship among the activities of T antigen involved in transforming cells, in binding to p53, and in enhancing the p53 cellular level. Moreover, this set of activities appears to be separable from the replicative ability of T antigen, based on the observation that 115K super T antigen is markedly defective for initiating viral DNA synthesis.     

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.     

Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen,bromodeoxyuridine and DNA

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (P<0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

SV40 Tag-specific cytotoxic T lymphocytes generated from the peripheral blood of malignant pleural mesothelioma patients

Malignant pleural mesothelioma (MPM) is an aggressive cancer, with survival of less than one year following diagnosis and treatment with current protocols. Recent studies have demonstrated the presence of the simian virus 40 (SV40)-like, large tumor antigen (Tag) in nearly 60% of MPMs. SV40 Tag is a viral-encoded tumor-specific antigen, and thus a potential target for the induction of anti-tumor immunity and the development of therapeutic vaccines. We describe here evidence for the existence of SV40 Tag-specific immune responses in patients with MPM whose tumors express Tag. Humoral immunity was demonstrated by the detection of IgG titers against Tag in serum samples from 1/3 of patients examined. CTLs were generated from the peripheral blood of an HLA-A2(+) MPM patient with a synthetic peptide representing an HLA-A2 binding epitope in SV40 Tag. The CTLs demonstrated epitope fine specificity, in that other peptides from SV40 Tag and a peptide from influenza virus were not recognized in the context of HLA-A2. Moreover, the CTLs were capable of recognizing mesothelioma tumor cells that expressed SV40 Tag, in an MHC class I restricted manner.     

Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system

DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively.     

Large T antigen-rich viral DNA replication loci in SV40-infected monkey kidney cells

The nuclear distribution of the large T antigen (T-Ag) during lytic infection of CV1 monkey kidney cells with SV40 virus was studied by immunoelectron microscopy. The viral protein was associated with the cellular chromatin and also accumulated within a small number of clearly delimited areas of the nucleoplasm. These T-Ag-rich areas were devoid of viral particles but contain 3-10 nm DNA filaments in an amorphous matrix. We have named these areas 'viral DNA/T-Ag loci.' The combination of the immunostaining for T-Ag with ultrastructural autoradiography revealed that these viral DNA/T-Ag loci were the sites of active SV40 DNA synthesis. We suggest that the viral DNA/T-Ag loci may represent definite structural domains specifically involved in viral DNA replication regulated by SV40-T antigen.     

Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA

Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically ( Fanning et al., 1981), sedimented at 5-6S and 14-16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end-labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5-6S and 14-16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35-bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.