The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication.

UNLABELLED: Merkel cell polyomavirus (MCPyV) plays an important role in Merkel cell carcinoma (MCC). MCPyV small T (sT) antigen has emerged as the key oncogenic driver in MCC carcinogenesis. It has also been shown to promote MCPyV LT-mediated replication by stabilizing LT. The importance of MCPyV sT led us to investigate sT functions and to identify potential ways to target this protein. We discovered that MCPyV sT purified from bacteria contains iron-sulfur (Fe/S) clusters. Electron paramagnetic resonance analysis showed that MCPyV sT coordinates a [2Fe-2S] and a [4Fe-4S] cluster. We also observed phenotypic conservation of Fe/S coordination in the sTs of other polyomaviruses. Since Fe/S clusters are critical cofactors in many nucleic acid processing enzymes involved in DNA unwinding and polymerization, our results suggested the hypothesis that MCPyV sT might be directly involved in viral replication. Indeed, we demonstrated that MCPyV sT enhances LT-mediated replication in a manner that is independent of its previously reported ability to stabilize LT. MCPyV sT translocates to nuclear foci containing actively replicating viral DNA, supporting a direct role for sT in promoting viral replication. Mutations of Fe/S cluster-coordinating cysteines in MCPyV sT abolish its ability to stimulate viral replication. Moreover, treatment with cidofovir, a potent antiviral agent, robustly inhibits the sT-mediated enhancement of MCPyV replication but has little effect on the basal viral replication driven by LT alone. This finding further indicates that MCPyV sT plays a direct role in stimulating viral DNA replication and introduces cidofovir as a possible drug for controlling MCPyV infection. IMPORTANCE: MCPyV is associated with a highly aggressive form of skin cancer in humans. Epidemiological surveys for MCPyV seropositivity and sequencing analyses of healthy human skin suggest that MCPyV may represent a common component of the human skin microbial flora. However, much of the biology of the virus and its oncogenic ability remain to be investigated. In this report, we identify MCPyV sT as a novel Fe/S cluster protein and show that conserved cysteine clusters are important for sT's ability to enhance viral replication. Moreover, we show that sT sensitizes MCPyV replication to cidofovir inhibition. The discovery of Fe/S clusters in MCPyV sT opens new avenues to the study of the structure and functionality of this protein. Moreover, this study supports the notion that sT is a potential drug target for dampening MCPyV infection.

Prevalence of JC polyomavirus large T antigen sequences among Iranian patients with central nervous system tumors.

The human neurotropic JC virus (JCV) is of significant interest due to its experimental neuro- oncogenic potential. In clinical samples from human central nervous system (CNS) tumors, detection of JCV sequences suggests a possible association with CNS neoplasms, but the results are discrepant worldwide. To assess the prevalence of JCV sequences in Iranian patients with primary and metastatic CNS malignancies, a total of 58 fresh CNS tumors were examined by quantitative real-time PCR targeting the JCV large T antigen (LT-Ag) gene, and JCV DNA load was determined as viral copy number per cell. All patients were immunocompetent, and none of them had received immunosuppressive therapy before surgical operation. JC virus LT-Ag sequences were found in a total of 15 (25.9 %) out of the 58 tested samples. In primary CNS tumors, JCV sequences were identified more frequently in meningiomas (50.0 %) and schwannomas (35.7 %). In metastatic CNS tumors, JCV LT-Ag was identified in one case with brain adenocarcinoma originating from lung cancer. No statistically significant association between JCV positivity and various types of CNS malignancies was observed (P = 0.565). The mean JCV LT-Ag copy number in 15 positive cases was 1.8 × 10(-4) ± 4.5 × 10(-4) copies per cell (range 1.0 × 10(-5)-1.78 × 10(-3) copies per cell). An inverse correlation between white blood cell (WBC) count and JCV copy number was observed, but this correlation was not statistically significant (R = -0.198, P = 0.480). This study provides the first data on the prevalence of JCV in primary and metastatic CNS tumors from Iranian patients.

The presence of JC virus in gastric carcinomas correlates with patient's age, intestinal histological type and aberrant methylation of tumor suppressor genes.

JC virus (JCV) is a neurotropic polyomavirus and the causative agent of progressive multifocal leukoencephalopathy. A role for JCV in gastrointestinal malignancies has been recently suggested. This study was carried out to determine the prevalence of polyomaviruses including JCV, BKV and SV40 in gastric cancers in Tunisia and to determine the clinicopathological characteristics of virus-associated gastric carcinomas. The presence of polyomaviruses DNA sequences was surveyed in 61 cases of primary gastric carcinomas and in 53 paired non-tumor gastric mucosa by PCR. Findings were correlated to clinicopathological parameters, p53 expression and methylation status of 11 tumor-related genes. Using PCR assays, JCV T-antigen sequence was more frequently detected in gastric carcinomas than in non-tumor gastric mucosa (26 vs 6%, P=0.03), while those of SV40 and BKV were not detected in any cases. Correlation analysis showed that JCV had higher frequency in patients older than 55 years (P=0.034) and in the intestinal histological type (P=0.04). With regard to methylation status, P16 and P14 showed significantly higher methylation frequencies in JCV-positive gastric carcinomas than in JCV-negative cases (P=0.007 and P=0.003, respectively). Moreover, the mean of the methylation index was significantly higher in JCV-positive than in JCV-negative cases (P=0.024). In multivariate logistic regression analysis, age of patients and the methylation index are only the two independent factors associated with JCV infection. Kaplan-Meier survival analysis showed a trend toward better survival for JCV-associated gastric carcinomas patients (log-rank, P=0.11). Our study suggests a role of JCV as cofactor in the pathogenesis of the intestinal type of gastric carcinomas in older persons.

Detection of Merkel cell polyomavirus DNA in Merkel cell carcinomas.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive tumour for which an increasing incidence has been reported. A new human polyomavirus, Merkel cell polyomavirus (MCV), was recently isolated from these tumours by applying digital transcriptome subtraction methodology. OBJECTIVES: To detect the presence or absence of MCV in MCCs and other, randomly selected neoplasms. METHODS: Nine primary or recurrent MCCs from seven patients were examined; 29 other tumours (squamous cell, basal cell and basosquamous carcinomas and malignant melanomas) were examined for comparative purposes. Viral large T protein (LT1 and LT3), and viral capsid protein (VP1) were detected by primer-directed amplification, using a polymerase chain reaction (PCR)-based method, and the amplified PCR products were analysed by agarose gel electrophoresis and subsequent sequence analysis. RESULTS: The presence of viral T antigen and/or viral capsid DNA sequences was demonstrated in seven of the eight MCC lesions. None of the comparative samples contained MCV DNA. CONCLUSIONS: Our findings strongly support the hypothesis that MCV infection may well be specific for MCC, and MCV may play a role in the pathogenesis of MCC.

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.

Murine tumor antigens: is it worth the search?

Standardized techniques that allow the direct identification of tumor antigens are now available. Several murine antigens recognized by T cells have already been identified. So far, the majority of these antigens derive from cellular proteins similar to those that give rise to human tumor antigens. While many of the known human tumor antigens are widely shared, most of the murine tumor antigens appear to be unique to the individual tumor from which they were isolated. Nonetheless, common features between murine and human tumor antigens are emerging, suggesting that these murine antigens will provide essential tools in the evaluation of antigen-based vaccines for the future treatment of cancer.

Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems.

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.

Extraction of SV40 T-antigen from lysates of transformed cells.

Subcellular fractions from SV-40 transformed hamster lens cells, prepared by chemical extractions, were tested for the presence of T-antigen by immunoautoradiography. Most of the T-antigen was present in the nucleus and was resistant to extraction by 2 M NaCl, indicating an association with the nuclear matrix. Another part of the T-antigen was, under certain conditions, resistant to extraction of the cells with a nonionic detergent. This T-antigen could be solubilized by Ca2+ at low temperature, conditions that also cause a specific depolymerization of microtubules.

Characterization of a novel Burkitt's lymphoma-associated antigen: GP70. Reactivity of anti-GP70 antibodies with various malignant and non-malignant cell lines.

A glycoprotein of 70 kDa (GP70) was isolated from sera of Burkitt's lymphoma (BL) patients and used to immunize rabbits. Anti-GP70 antibodies at a high titer were obtained and used for screening of cancer cells of various origin by the indirect immunofluorescence test. Thus, 66% of BL-cell lines tested were positive to GP70. On the other hand, all lymphoblastoid cell lines tested were negative. Moreover, all peripheral blood cells and mononuclear cells from tonsils were negative, indicating specificity of antibodies to malignant transformation. Comparison between positively stained BL-cell lines indicated no correlation between the presence of GP70 and EBNA. Positive stain (1-5%) obtained with bone marrow cells might indicate that anti-GP70 antibodies are directed against a surface membrane differentiation glycoprotein.

A comparison of methods for the extraction of nucleoprotein complexes from SV40-infected cells.

A comparison was made between two alternative methods for the nuclear extraction of Simian virus 40 (SV40) virions and nucleoprotein complexes (NPCs) from SV40-infected TC7 cells. The low-salt hypotonic method of Su and DePamphilis (1976) was compared with the detergent method of Garber et al. (1978), since other methods had been shown to result in virion breakdown. There was no disruption of mature SV40 virions with either of these extraction procedures. There was, however, considerably more effective extraction of SV40 NPCs, known to contain large tumor (T) antigen, using the low-salt hypotonic method as opposed to the detergent method. Thus, the low-salt hypotonic method for extraction should be the method of choice when studying SV40 DNA replication or the function of SV40 T antigen in SV40 nucleoprotein complexes.

Breast cancer and T-cell-mediated immunity to proteins of the mouse mammary tumour virus (MMTV).

We have previously reported that breast cancer patients and some healthy subjects show positive T-cell-mediated immune responses to a semi-purified mouse mammary tumour viral pool (MMTV). We have now used Western blotting to analyse the specificity of the response and to determine the target polypeptides. Two types of T-cell response to the viral antigens were examined, proliferation and MIF release, the latter implies a DTH status in vivo where primed lymphocytes are involved. Two viral fractions were used, one containing a glycoprotein, the 52 kD major virus envelope, and the other containing the 28 kD main virus core protein. We analysed both patients and healthy subjects whose T-cells proliferated to the MMTV total extract (viral pool). The T-cell response in the patients was shown to be viral specific since both the T-cell proliferation (21/25) and MIF release (17/19) were directed against viral components of the pool (gp 52 and/or p 28). The T-cell response in the healthy control subjects was shown to be mostly directed against a species-specific albumin component of the extract. In addition, the monocyte integrity required for the MIF response was altered in the breast cancer patients. The monocytes from one patient out of three failed to respond to MIF, even though the lymphokine was released normally by the patients' activated T-cells.

Epstein-Barr virus (EBV) hypersensitivity of peripheral B lymphocytes in patients with EBV genome-positive Burkitt's lymphoma.

Peripheral blood mononuclear cells (PBMC) from four Japanese patients with Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) during remission were exposed to the B95-8 strain of EBV. Maximum concentrations of the EBV-determined nuclear antigen (EBNA) before cellular DNA synthesis were similar to those of healthy counterparts. Subsequently, EBV-immortalised cell lines were established. These immortalised lymphoblastoid cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinfected with the P3HR-1 strain of EBV. EBV early antigens (EA) and viral capsid antigen (VCA) were expressed in approximately 3-10 fold higher concentrations by these lymphoblastoid cells than by those from patients with other types of malignant neoplasia including EBV genome-negative BL and from healthy counterparts. Moderate to extremely high IgG antibody titres to EBV VCA as well as IgG antibodies to EA were demonstrated in these patients during the study. These results suggest that defective underlying cellular mechanisms for regulating the replication of EBV may be present in patients with EBV genome-positive BL.

Relationship between the tumour-associated antigen 90K and cytokines in the circulation of persons infected with human immunodeficiency virus.

A tumour-associated antigen known as 90K has been found in high concentrations in the serum of patients infected with human immunodeficiency virus (HIV) even in the absence of neoplastic complications. In order to investigate the relationship between the production of 90K and soluble inflammatory mediators, we studied serum concentrations of the antigen, tumour necrosis factor-alpha (TNF-alpha), interleukin-I-alpha (IL-I-alpha), interferon-gamma (IFN-gamma), IFN-alpha, neopterin and beta 2-microglobulin (beta 2-m) in patients with non-neoplastic HIV infection at various stages of disease and in control persons. The antigen was detected in all those studied but its concentration was higher in HIV-infected patients compared with controls (P < 0.001), increasing progressively with advancing stages of disease. There was a negative correlation between concentrations of 90K and IL-I-alpha in patients in U.S.A. Centers for Disease Control groups II and III (P < 0.02) and also between that of 90K and both TNF-alpha (P < 0.01) and IL-I-alpha (P < 0.05) in control persons. The results indicate that 90K is not merely a tumour-associated antigen and that its production may be part of immune and inflammatory responses in the absence of neoplasia. The correlation between the concentrations of 90K and of some cytokines in asymptomatic patients and healthy persons suggests that 90K may be part of a network of immune and inflammatory reactants.

Partial purification of Simian virus 40 large T antigen by immunoaffinity chromatography and its detection by enzyme-linked immunosorbent assay.

Simian virus 40 (SV40) large T antigen was partially purified from small amounts of SV40-infected and SV40-transformed cells by immunoaffinity chromatography with high recovery. T antigen, in both crude and partially purified states, was detected rapidly by a sensitive and quantitative enzyme-linked immunosorbent assay (ELISA). Stability of the partially purified T antigen was found to increase by addition of 0.01% bovine serum albumin (BSA).

[Isolation and characteristics of the fibroblast cell line of rats cotransformed by the DNA of simian adenovirus SA7 (C8) and hepatitis B virus]. / Poluchenie i kharakteristika linii kletok fibroblastov krysy, kotransformirovannykh DNK adenovirusa obez'ian SA7 (C8) i virusa gepatita B.

Transformed cell line (SH2) was established by means of co-transfection of primary rat embryonal fibroblasts by DNA of high-oncogenic simian adenovirus SA7 (C-8) and hepatitis B virus. SH2 cells possess a transformed phenotype and high oncogenicity both for allogenic (rats) and xenogenic (hamsters) animals. 100 SH2 cells induce tumours in newborn (3 day) hamsters. 10(4) SH2 cells inoculated to adult hamsters induce tumours in approximately 50% cases on the 20th-30th day. Possible mechanisms of significant stimulation of SH2 cells oncogenic properties after co-transfection by DNA of oncogenic adenovirus SA7 and hepatitis B virus are discussed.

Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens.

Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125I. The 125I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography.