Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.     

Lack of protein expression of the Simian virus 40 large T antigen in human lymphomas

Several studies have detected Simian virus 40 (SV40) deoxyribonucleic acid sequences in human tumor tissues, including lymphomas, mainly by the polymerase chain reaction, but these data were not confirmed by subsequent investigations. Regional differences in the distribution of the SV40 and/or technical difficulties have been taken into account to explain these divergent results, but because only a few such studies dealt with the expression of SV40 proteins in tumor tissues, we investigated the expression of the SV40 large T antigen in human lymphomas by immunohistochemistry. Tissue microarrays containing Non-Hodgkin's-lymphomas and Hodgkin's-lymphomas were constructed utilizing archival samples encompassing the years 1974--2001 from Italian, Swiss and Austrian patients. Expression of the SV40 large T antigen was analysed by highly specific and sensitive immunohistochemistry using a mouse monoclonal antibody. Protein expression of the large T antigen was not detected in 655 Non-Hodgkin's-lymphomas or in 337 Hodgkin's- lymphomas. The results suggest the absence of an association between SV40 large T antigen and human lymphomas.     

Simian virus 40 in posttransplant lymphoproliferative disorders

Simian virus 40 (SV40) is an oncogenic DNA virus, which is an emergent pathogen implicated in some human malignancies, including B-cell lymphomas. As with other malignancies that occur during immunosuppression, it is hypothesized that SV40 infections may be associated with some posttransplant lymphoproliferative disorders (PTLDs). Specimens were tested initially for Epstein-Barr virus (EBV) by in situ hybridization for EBV-encoded small RNA and/or by immunohistochemical staining for EBV-latent membrane protein 1. Coded DNA specimens extracted from formalin-fixed, paraffin-embedded tissues from 22 PTLD cases were examined by polymerase chain reaction using primers for the SV40 large tumor antigen (T-ag) gene and confirmed by sequence analysis. In addition, samples were assessed for the expression of SV40 T-ag by immunohistochemical staining. Epstein-Barr virus was detected in 18 (82%) of 22 PTLD cases. Simian virus 40 T-ag sequences were detected in 2 (13%) of the 16 cases with amplifiable DNA: one with EBV-negative T-cell PTLD and the other with EBV-positive B-cell monomorphic PTLD. Immunohistochemical staining showed expression of SV40 T-ag in 1 of 2 cases containing viral DNA sequences and in none of the SV40 T-ag DNA-negative samples. Expression of SV40 T-ag was restricted to malignant cells and not to reactive lymphocytes. These results suggest that SV40 may be associated with a small subset of PTLD cases. Additional studies are needed to determine the role of SV40 in EBV-negative PTLD.     

猴空泡病毒40核酸序列检测法的优化研究

目的 对通常使用的猴空泡病毒40(Simian vacuolating virus 40,SV40)核酸序列检测法进行优化,寻找敏感性高、特异性强、适用面广的SV40核酸序列检测引物.方法 以21个SV40毒株完全基因组为基础数据,用Primer Premier 5.00软件重新设计两对SV40 DNA检测引物,用Oligo 6.71软件和DNAMAN 6.0.40软件对引物参数进行分析,将分析结果与通常使用的检测引物进行比较.用不同稀释度SV40核酸序列作模板,比较4对引物检测的敏感性.分别用无菌水、Vero细胞DNA、SV40 DNA作模板检测4对引物的特异性.结果 对于21个SV40病毒株,优化引物对VP1和T的序列是保守的;对于接受号为J02400、NC_001669、AF316139和AF316141的4个病毒株,通常使用的引物对GCVP1和GCT的序列是保守的;用同一稀释度的SV40 DNA作模板,引物对VP1和T的扩增效率明显高于引物对GCVP1和GCT;在特异性检测比较中,引物对VP1和T没有出现非特异性扩增条带,引物对GCVP1和GCT在100 bp处出现非特异性扩增条带.结论 优化的SV40核酸序列检测法具有敏感性高、特异性强、检测面广、引物及其PCR产物序列保守等特点.     

Simian virus 40 large T antigen isoelectric focuses as multiple species with varying phosphate content.

Simian Virus 40 large T antigen (TAg), radiolabeled with [³2P]orthophosphate and with/without [³H]methionine, was found to isoelectric focus as four distinct species distributed in a highly reproducible pattern. These species have been found to differ in their ${}^{32}\text{P}{}^3\text{H}$ ratios indicating that individual monomers of large TAg are heterogeneous in their phosphate content and suggesting, further, that large TAg may be differentially phosphorylated in vivo. Also, by increasing the concentration of large TAg the more basic species can be displaced into the more acidic species suggesting that the different isoelectric focusing forms represent aggregates of large TAg with varying phosphate content.     

Simian virus 40 infection in humans and association with human diseases: results and hypotheses

Simian virus 40 (SV40) is a monkey virus that was introduced in the human population by contaminated poliovaccines, produced in SV40-infected monkey cells, between 1955 and 1963. Epidemiological evidence now suggests that SV40 may be contagiously transmitted in humans by horizontal infection, independent of the earlier administration of SV40-contaminated poliovaccines. This evidence includes detection of SV40 DNA sequences in human tissues and of SV40 antibodies in human sera, as well as rescue of infectious SV40 from a human tumor. Detection of SV40 DNA sequences in blood and sperm and of SV40 virions in sewage points to the hematic, sexual, and orofecal routes as means of virus transmission in humans. The site of latent infection in humans is not known, but the presence of SV40 in urine suggests the kidney as a possible site of latency, as it occurs in the natural monkey host. SV40 in humans is associated with inflammatory kidney diseases and with specific tumor types: mesothelioma, lymphoma, brain, and bone. These human tumors correspond to the neoplasms that are induced by SV40 experimental inoculation in rodents and by generation of transgenic mice with the SV40 early region gene directed by its own early promoter-enhancer. The mechanisms of SV40 tumorigenesis in humans are related to the properties of the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag). Tag acts mainly by blocking the functions of p53 and RB tumor suppressor proteins, as well as by inducing chromosomal aberrations in the host cell. These chromosome alterations may hit genes important in oncogenesis and generate genetic instability in tumor cells. The clastogenic activity of Tag, which fixes the chromosome damage in the infected cells, may explain the low viral load in SV40-positive human tumors and the observation that Tag is expressed only in a fraction of tumor cells. "Hit and run" seems the most plausible mechanism to support this situation. The small tag, like large Tag, displays several functions, but its principal role in transformation is to bind the protein phosphatase PP2A. This leads to constitutive activation of the Wnt pathway, resulting in continuous cell proliferation. The possibility that SV40 is implicated as a cofactor in the etiology of some human tumors has stimulated the preparation of a vaccine against the large Tag. Such a vaccine may represent in the future a useful immunoprophylactic and immunotherapeutic intervention against human tumors associated with SV40.     

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.     

SV40 large T antigen-specific human T cell memory responses

The continued presence of simian virus 40 (SV40), a monkey polyomavirus, in man is confirmed by the regular detection of SV40-specific antibodies in 5-10% of children who are unlikely to have received contaminated polio-vaccines. The aim of our experiments was to find cellular immunological evidence of SV40 infection in humans by testing memory T cell responses to SV40 large T antigen (Tag). As there is some indication that the virus may be present in malignant pleural mesothelioma (MPM) cells, we analyzed T cell responses in MPM patients and in healthy donors. The frequencies of responding T cells to overlapping Tag peptides were tested by cytokine flow cytometry. CD8+ T cells from 4 of 32 MPM patients responded (above twofold of control) to SV40 Tag peptides, while no positive responses were detected in 12 healthy donors. Within SV40 Tag we identified three 15 amino acid-long immunogenic sequences and one 9 amino acid-long T cell epitope (p138) (138FPSELLSFL146), the latter including a HLA-B7-restriction motif. T cell responses to p138 were SV40-specific as T cells stimulated with p138 did not cross-react with the corresponding sequences of Tag of human polyomaviruses BKV and JCV. Similarly, the relevant BKV and JCV Tag peptides did not generate T cell responses against SV40 TAg p138. Peptide-stimulated T cells also killed SV40 Tag-transfected target cells. This article demonstrates the presence, and provides a detailed analysis, of SV40-specific T cell memory in man.     

Transformation of rat cell by SV40 virus

By analysis of data on the transformation of rat cells by a wild type SV40 virus (SV3T3)-1 at the temperature of 33 degrees C and 39 degrees C, I conclude that the transformation protein could be a complex of viral and host proteins.     

The carboxyl-terminal domain of large T antigen rescues SV40 host range activity in trans independent of acetylation

The host range activity of SV40 has been described as the inability of mutant viruses with deletions in the C terminal region of large T Ag to replicate in certain types of African green monkey kidney cells. We constructed new mutant viruses expressing truncated T Ag proteins and found that these mutant viruses exhibited the host range phenotype. The host range phenotype was independent of acetylation of T Ag at lysine 697. Co-expression of the C terminal domain of T Ag (aa 627-708) in trans increased both T Ag and VP1 mRNA as well as protein levels for host range mutant viruses in the restrictive cell type. In addition, the T Ag 627-708 fragment promoted the productive lytic infection of host range mutant viruses in the nonpermissive cell type. The carboxyl-terminal region of T Ag contains a biological function essential for the SV40 viral life cycle.     

人脑肿瘤组织中SV40感染及其临床意义

目的 探讨猴病毒40（SV40）与人脑肿瘤发病的关系。方法 采用聚 一等奖反应（PCR）和原杂交（ISH）同时检测30例正常人脑组织和198例人及脑肿瘤组织以及SHG44和BT3 25两株人脑胶质瘤细胞系中SV40 DNA充列;并对SV40 DNA阳性肿瘤细胞采用免疫共沉淀和Western blot检测大T抗原（Tag）的表达及Tag-RB复合物的存在。结果 人脑肿瘤组织PCR SV40 DNA阳性率为48.5％（96／198）,其中胶质瘤47.2％（42／89）,脑膜瘤48.8％（21／43）,脑垂体腺瘤51. 4%(18/35),神经鞘瘤43.8％（7／16）,先天性肿瘤53.3％（8／15）,正常人脑组织PCR SV40 DNA阳性率为6.7％（2／30）。SHG44及BT325两株细胞中也分别检测出SV40的DNA序列。人脑肿瘤组织SV40 DNA阳性率显著高于正常人脑组织（P＜0.01）。经ISH,仅在96例PCR SV40 DNA阳性的及脑肿瘤组织中检出87例阳性,2例SV40 DNA阳性的正常人脑组织中1例ISH阳性,SHG44及BT325两株细胞ISH SV40 DNA均阳性。SV40 DNA定位于肿瘤细胞核,阳性细胞呈弥温或片灶状分布。96人列SV40 DNA阳性脑瘤组织Tag表达阳性75例,所有Tag表达阳性瘤组织均发现Tag与RB形成特异性复合物。结论 人脑肿瘤组织中存在SV40感染,提示SV40感染与有脑肿瘤有关;在人脑肿瘤组织中Tag广泛表达,Tag可能是SV40在有禽肿瘤发生发展中起作用的重要因素,S V40 Tag与RB形成特异性复合物Tag-RB,导致RB活性,有是SV40致人脑肿瘤发生的一个重要机理。     

Sensitive two-site sandwich enzyme immunoassay with antipeptide antibodies for detection of viral antigens

Two-site sandwich ELISAs with antipeptide antibodies were developed for measuring the amounts of polyoma virus medium T and SV40 virus large T antigen in extracts from virus infected and transformed cells. In order to exclude crossreactive proteins, two antipeptide antibodies directed against different regions of the antigen were used in series. Both antigenic determinants have to be recognized for the assay to score positive. A positive signal was obtained with as little as 10 μ1 of polyoma virus infected cell lysate and 100 μl of extract prepared from medium T antigen-transformed FR18-1 cells which were shown to contain 37 and 15 pg of medium T antigen, respectively. This two-site sandwich ELISA is a sensitive tool to detect novel or closely related proteins.     

Quantitative analysis of the immunogenic activity of SV40 virus and of tumor cells induced by this virus

A study of specific antitumor immunity created in Syrian hamsters by oncogenic virus SV40 and by tumor cells induced by the same virus showed that the level of specific resistance depends on the immunizing dose of virus and cells. Investigation of the resistance-inducing activity of a wild strain of SV40 virus showed that the minimal dose inducing resistance in hamsters was ten times higher than for the tsA-30 mutant of the virus. The minimal resistance-inducing dose of irradiated cells of a tumor induced by the same strain of SV40 virus was 9·10⁵ cells; a tenfold increase in the dose led to a significant increase in the level of specific antitumor immunity.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 8, pp. 223–225, August, 1978.     

Temperature Sensitive Simian Virus 40 Large T Antigen Immortalization of Murine Odontoblast Cell Cultures: Establishment of Clonal Odontoblast Cell Line

During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchy-mal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 × 10⁵well) were plated as monolayers and grown in α-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 μg/ml ascorbic acid. Cultures were maintained for 6 days at 37°C in a humidified atmosphere of 95% air and 5% CO₂ with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 μg/ml of polybrene, the media was replaced with selection media containing 300 μg/ml of G418, and the cultures incubated at 33°C for one month with media changes every 3–5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33°C. One established cell line, M06-G3, showed high constitutive expression of dentin phosphoprotein, type I collagen and alkaline phosphatase, characteristic of an odontoblast phenotype. These results demonstrate the establishment of a stable murine odontoblast cell line which retains the tissue-specific expression for a number of DECM proteins.     

A method for transforming human liver epithelial cells by transfection using a plasmid containing SV40 early region gene

Summary A method is described for establishing continuous cell lines of liver epithelial cells by transformation of cultured hepatocytes isolated by collagenase/dispase of adult human liver tissue. Between 2 to 5×10⁵ hepatocytes are inoculated into a 60 mm culture dish. The cells are incubated in a serum-free medium. Once the cells begin to divide, they are transformed by transfection with a plasmid containing SV40 early region genes.     

新城疫病毒HN基因对肿瘤抗原诱导的抗肿瘤免疫的增强作用

目的 了解新城疫病毒HN基因增强肿瘤抗原诱发的抗肿瘤免疫反应的作用。并对其作用机制进行探讨。方法 将构建的HN基因-癌胚抗原（CEA）cDNA共表达质粒（pcD-CEA/HN)免疫小鼠,通过淋巴细胞增殖实验,NK细胞活性检测了解HN对抗CEA免疫反应的影响,并以免疫组化的手段追踪HN质粒表达产物在体内组织的分布,以及HN质粒对荷瘤小鼠肿瘤生长,抗肿瘤免疫反应的影响。结果 （1）共表达质粒pcD-CEA/HN免疫小鼠获得了较其他对照组强的淋巴细胞增殖指数及NK细胞活性。（2）HN质粒在肌肉,肿瘤组织有明显的表达。（3）HN质粒对肿瘤长生具有一定的抑制作用。并能增强荷瘤小鼠的抗肿瘤免疫。结论 新城疫病毒HN基因对肿瘤抗原诱导的抗肿瘤免疫具有增强作用。     

Absence of simian virus 40 DNA sequences in primary central nervous system lymphoma in HIV-negative patients

Simian virus 40 (SV40) is known to induce primary brain tumors and lymphomas in animal models. Recently, it was also associated with the pathogenesis of human non-Hodgkins lymphomas. In the present study, we investigated primary central nervous system lymphomas (PCNSL), a defined subgroup of diffuse large B-cell lymphoma confined to the central nervous system, for the presence of SV40 DNA. Frozen tissue samples of 23 PCNSL derived from human immunodeficiency virus-negative patients were analyzed by two different, fully nested polymerase chain reaction protocols. SV40 DNA sequences could not be detected in any of these samples. Thus, SV40 can be added to the list of viruses that have already been excluded as pathogenetically relevant cofactors in PCNSL.     

Immortalization of human fibroblasts by liposome-mediated transfer of SV40 early region genes

Transfer of simian virus 40 (SV40) early region genes into normal cells is one of the most useful methods of obtaining immortalized cell lines. The SV40 early region encodes viral proteins that cause an increased, but finite, cellular proliferative potential; immortalization requires additional genetic changes. The following protocol describes plasmid-mediated transfer of SV40 early region genes into human fibroblasts and cell culture conditions designed to maximize the probability of obtaining an immortalized cell line. This approach may be used for any cell type.