Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40␣T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model. Received: 20 August 1998 / Accepted: 25 November 1998     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants

Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National Institutes of Health, Bethesda, MD.     

Haploid male germ cells show no susceptibility to transformation by simian virus 40 large tumour antigen in transgenic mice.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40 (simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸。不同分离株bp数略有差异。SV40病毒为强DNA肿瘤病毒,具有使啮齿类动物及人源多种组织培养细胞永生化和转化能力。SV40病毒早期基因编码两个早期非结构蛋白即小T抗原(ST-ag、)和大T抗原(LT-ag),与病毒诱导的肿瘤发生有关。近年来研究表明,从猴体组织新分离的SV40株与实验室参考株SV40-776及SV40-B株相比较,具有明显的遗传异质性,并且SV40遗传变     

Transcriptionally regulated immortalization overcomes side effects of temperature-sensitive SV40 large T antigen

The temperature-sensitive mutant of the SV40 virus large T antigen (TAg) tsA58 is frequently employed for the conditional immortalization of primary cells. By increasing the temperature to 39 degrees C, the activity of the mutant TAg is reduced and the status of such cells may then resemble more closely that of primary cells. As an alternative, we used a novel immortalization vector with a tetracycline-regulated expression of the wild-type TAg. This enabled us to investigate the effects of the immortalizing gene expression and of temperature shifts independently of each other. Even for wild-type TAg-derived cell lines the elevated temperatures led to various clone-dependent phenotypes. This suggests that in freshly established cell lines temperature-sensitive growth phenotypes can arise spontaneously and independently of a temperature-sensitive immortalizing gene. Similar effects were observed with spontaneously immortalized cells. On the other hand, not all of the ts-TAg-derived cell lines were proliferation arrested at the non-permissive temperature. Therefore, the assumption that temperature-sensitive growth is solely due to the ts-TAg must be verified for each ts-TAg-derived cell line individually. This complexity could be avoided by using the autoregulatory immortalization vector expressing the wild-type TAg.     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40(simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸,不同分离株bp数略有差异.     

表达EB病毒主要膜蛋白的非复制型重组痘苗病毒毒力及体液免疫反应

利用表达EBV GP350/220的非复制型重组痘苗病毒VMA△CK及复制性重组痘苗病毒VMA,注射乳鼠脑内,观察毒力。同时,用VMA△CK及VMA免疫Bal/c小鼠,经ELISA测定其特异性抗体水平。免疫血清用抗体中和EBV感染Raji细胞抑制产生早期抗原（NEA）法测定其中和抗体滴度。将免疫血清与P3HR-1细胞共同孵育后,测上清中EBV滴度以观察抗体体制EBV从P3HR-1细胞中释放效应。结果发现,VMA△CK毒力明显低于VMA,其LD50为4.5PFU/0.02ml,而VMA△CK的LD50大于10^6PFU/0.02ml。VMA△CK与VAM免疫血清中抗GP350/220抗体水平无明显差异,而初兔后抗痘苗病毒抗体水平VMA免疫组较VMA△CK免疫组高5倍左右。VMA△CK免疫组血清中和抗体水平没有明显差异。VMA△CK免疫血清稀释度与P3HR-1细胞培养上清中EBV滴度有剂量依赖关系。     

Antigenic structure of simian virus 40 large tumor antigen and association with cellular protein p53 on the surfaces of simian virus 40-infected and -transformed cells.

The antigenic structure of simian virus 40 (SV40) large tumor antigen (T-ag) in the plasma membranes of SV40-transformed mouse cells and SV40-infected monkey cells was characterized as a step toward defining possible biological function(s). Wild-type SV40, as well as a deletion mutant of SV40 (dl1263) which codes for a truncated T-ag with an altered carboxy terminus, was used to infect permissive cells. Members of a series of monoclonal antibodies directed against antigenic determinants on either the amino or the carboxy terminus of the T-ag polypeptide were able to precipitate surface T-ag (as well as nuclear T-ag) from both SV40-transformed and SV40-infected cells. Cellular protein p53 was coprecipitated with T-ag by all T-ag-reactive reagents from the surface and nucleus of SV40-transformed cells. In contrast, T-ag, but not T-ag-p53 complex, was recovered from the surface of SV40-infected cells. These results confirm that nuclear T-ag and surface T-ag are highly related molecules and that a complex of SV40 T-ag and p53 is present at the surface of SV40-transformed cells. Detectable levels of such a complex do not appear to be present on SV40-infected cells. Both the carboxy and amino termini of T-ag are exposed on the surfaces of SV40-transformed and -infected cells. The possible relevance of the presence of a T-ag-p53 complex on the surface of SV40-transformed cells and its absence from SV40-infected cells is considered.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.     

Umbilical cord endothelial cells expressing large T antigen: Comparison with primary cultures and effect of cell age

Summary A number of human endothelial cell lines from umbilical cord cells (HUVECs) have been generated by transfection with SV40 large T and small t antigen sequences. Comparison of these lines with primary cultures of HUVECs has been carried out by monitoring the expression of a number of endothelial cell markers with specific regard to cell age. The secreted levels of the protein plasminogen activator inhibitor (PAI) was found to be significantly reduced in SV40-transfected cells when compared to untransfected controls. Tissue plasminogen activator (tPA) and urokinase (uPA) levels were unchanged. As cells entered crisis, there was a rapid and significant increase in the levels of tPA, uPA, and PAI and this was observed for all clones screened. The endothelial cell marker von Willebrand Factor (vWF) was found intracellularly and was also secreted into the medium. The levels were not altered between transfected and untransfected cells. Angiotensin converting enzyme (ACE) activity was maintained in cell lines at levels found in nonimmortalized HUVECs. Both isoforms (α and β) of IL-1 (interleukin-1) increased as cells approached crisis, and the presence of these cytokines may be responsible for the increased levels of tPA, PAI, and uPA. With one exception, the ability of the transfected cells to produce prostacyclin (PGI₂) was lost by all clones.     

Intercellular communication and the control of growth: XII. Alteration of junctional permeability by simian virus 40. roles of the large and smallT antigens

Summary We studied the action of temperature-sensitive mutant simian virus 40—a transformation-inducing DNA virus—on the junctional permeability to mono-, di- and triglutamate in rat embryo-, pancreas islet (epithelia)-, and 10T1/2 cell cultures. Junctional permeability was reduced (reversibly) in the transformed state. To dissect the genetics of this alteration, we used two kinds of mutant virus DNA. One kind had a temperature-sensitive mutation on theA gene, rendering the largeT antigen (the gene product) thermolabile (T ⁺ T ^–). The other had a deletion on theF gene, in addition, abolishing (permanently) the expression of the littlet antigen (t ^–). The junctional alteration occurred in the conditionT ⁺ t ⁺, but not in the conditionsT ^– t ⁺,T ⁺ t ^– orT ^– t ^–. Both antigens, thus, are necessary for this junctional alteration—a genetic requirement identical to that for decontrol of growth (but distinct from that of the cytoskeletal alteration).     

Transient gene expression using valproic acid in combination with co-transfection of SV40 large T antigen and human p21CIP/p27KIP

Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. by co-expression of human cell cycle inhibitors p21^CIP/p27^KIP and Simian virus 40 large T antigen (SVLT). In this study, we investigated the effect of this enhancer protein complex on the TGE experiments in a cell-cycle arrested condition of HEK293F cells induced by valproic acid. Growth profiles, consumptions of nutrients, formations of waste products, and product titers of recombinant human antibodies (huAb) were monitored during the 7-day cultivation time. Our results showed that the use of enhancer proteins increased the product yields in a growth arrest condition as well. During the growth phase, no differences were detected regarding viable cell densities (VCDs), viabilities, growth rates, and cell diameters between the TGE experiments with and without enhancer proteins. However, during the declining phase VCD and viability showed slightly higher values at day 6 and 7 in the presence of enhancers. Furthermore, we could not detect any differences in glucose and glutamine metabolism during batch cultivations with co-expression of enhancer proteins. Taken together, the special complex of enhancer proteins did not contribute to further enhancement of growth arrest and shift in the main cell metabolisms, but resulted in higher cell viability during the decline phase. Our observations suggest that the human cell cycle inhibitors p21^CIP/p27^KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins.