Loss of heterozygosity on the short arm of chromosome 3 in nasopharyngeal carcinoma.

A consistent loss of constitutional heterozygosity within a specific chromosome locus in a tumor type is suggestive of a tumor suppressor gene important in the genesis of that tumor. We studied whether such genetic alterations are involved, in the development of nasopharyngeal carcinoma (NPC). Tumor and matched blood leukocytes DNA from eleven Hong Kong Chinese patients with primary NPC stages I to IV were subjected to restriction fragment length polymorphism (RFLP) analysis using chromosome 3-specific polymorphic probes. Such probes are assigned to chromosomal region 3p25 (RAF-1), 3p24-22.1 (ERBA beta), 3p21 (DNF15S2), 3p14 (D3S3), and 3q12 (D3S1). The breakpoint varied among tumors, ranging in extent from 3p21-14. However, 100% frequency of complete loss of heterozygosity was observed at two chromosomal loci: RAF-1 locus (ten of ten cases at 3p25) and D3S3 locus (nine of nine cases at 3p14), in all evaluable NPC patients, suggesting the presence of putative tumor suppressor gene(s) within or close to these defined regions. The observed consistent deletion of alleles on the short arm of chromosome 3 in the NPC cases, which is in line with our previously reported and present cytogenetic findings, may represent a critical event in the multistep genesis of NPC. The present report also identifies defined loci for linkage studies on NPC families.

Biochemical characterization of Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) was immunoprecipitated from latently Epstein-Barr virus-infected lymphocytes with a polyclonal serum raised against the EBNA-2A C terminus. The nucleus contained three subfractions of EBNA-2A which could be distinguished by their resistance to salt extraction: (i) a nucleoplasmatic fraction that was solubilized at 50 mM NaCl, (ii) a chromatin-associated fraction extractable at 1.5 M NaCl, and (iii) a nuclear matrix-associated fraction solubilized only by boiling with buffer containing 2% sodium dodecyl sulfate. The three subfractions were phosphorylated; it was demonstrated that the nucleoplasmatic and the chromatin-associated fractions were phosphorylated at serine and threonine residues. The half-life of the EBNA-2A protein was determined by cycloheximide treatment and by pulse-chase experiments and was found to be at least 24 h. The turnover of the phosphate residues bound to the two salt-soluble subfractions was determined to be approximately 6 to 9 h, suggesting a possible role of the phosphorylation in the regulation of the biological activity of EBNA-2A. Dephosphorylation of EBNA-2A resulted in an increased mobility of the protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and indicated the presence of differentially phosphorylated subclasses of the protein. Analysis of EBNA-2A by sucrose gradient centrifugation revealed the existence of two subclasses of complexed molecules which exhibited sedimentation coefficients of approximately 13S and 34S.

The BZLF1 protein of EBV has a coiled coil dimerisation domain without a heptad leucine repeat but with homology to the C/EBP leucine zipper.

The EBV transactivator protein BZLF1 can bind many sites in the EBV genome, most of which have homology to a consensus AP-1 site, the binding site for the fos/jun family of transcription factors. Here we present evidence that BZLF1 can also recognise the binding site for the CCAAT/enhancer binding protein C/EBP and that a BZLF1 binding site within the BZLF1 promoter is recognised by the C/EBP protein. Analysis of the BZLF1 DNA binding domain suggests that the BZLF1 protein binds to DNA as a dimer using sequences adjacent to a basic DNA binding motif. The BZLF1 dimerisation domain does not have a heptad repeat of leucine residues common to leucine zipper proteins but does have characteristics of a coiled coil structure, as judged by site directed mutagenesis. We therefore propose that the dimerisation domain of BZLF1 is structurally related to the coiled coil structure of leucine zippers but lacks the highly conserved leucine repeat. We show that the PZLF1 dimerisation domain has residues in common with the C/EBP leucine zipper and discuss the possible implications of this relationship.

Oral hairy leukoplakia in HIV infection: a diagnostic pitfall.

Twenty-nine human immunodeficiency virus (HIV)-infected patients with white, nonremovable lesions on the lateral border of the tongue, clinically suggestive of oral hairy leukoplakia (HL), were studied. In particular, the value of local antifungal therapy in establishing the diagnosis of HL was investigated. In 15 patients (52%) the lesions could be ultimately attributed to a candidal infection of the tongue. In 10 of the remaining 14 patients, a biopsy was obtained from lesions persisting after local antifungal treatment. In all biopsy specimens, the diagnosis of HL was confirmed by histopathologic examination and the demonstration of Epstein-Barr virus DNA by polymerase chain reaction, Southern blot hybridization, and DNA in situ hybridization. The present data confirm that the diagnosis of HL in HIV-infected patients cannot be reliably made on clinical criteria alone, but requires histopathologic confirmation including the demonstration of Epstein-Barr virus DNA, preferably by DNA in situ hybridization. However, with regard to the differential diagnosis of white, nonremovable lesions on the lateral border of the tongue in HIV-infected patients, the present study suggests that persistence of lesions after local antifungal therapy is highly suggestive of HL.

Oral melanotic macules in patients infected with human immunodeficiency virus.

A group of 217 patients seropositive for human immunodeficiency virus (HIV) were studied for 2 years, during which time pigmented lesions of the oral mucosa developed in 14 (6.4%) of them. The lesions were well circumscribed in some cases and diffuse in others. In some patients the macules enlarged or recurred after surgical excision. In two patients the macules appeared during the administration of zidovudine. Clinical and laboratory evidence of adrenal insufficiency was not detected in any of the patients examined. The histologic appearances were those of melanotic macules. No ultrastructural alterations of the melanocytes were observed. Two of these macules also contained Epstein-Barr virus, and in one case normal oral mucosa was examined and also contained Epstein-Barr virus in the epithelial cells. As a control group we examined 180 health care workers who did not belong to any risk category, and 30 intravenous drug abusers who tested seronegative to HIV. Oral melanotic pigmentation was found in eight of the control subjects (3.6%). The difference was not statistically significant (p = 0.3097). Our study shows that oral macules do not occur more frequently in HIV-infected patients. However, the clinical behavior of these lesions appears to be different during the course of HIV infection. In some HIV-infected patients the cause of the macules might relate to the administration of zidovudine and antifungal or antibacterial drugs. In others the cause remains unknown and could be due to multiple factors.

Diagnosis of oral hairy leukoplakia by ultrastructural examination of exfoliative cytologic specimens.

Lingual exfoliative cytologic specimens (scrapings) were obtained from 18 patients positive for human immunodeficiency virus with clinical oral hairy leukoplakia. Buccal mucosal scrapings were obtained from 12 of these patients. The specimens were processed for examination by transmission electron microscopy (TEM). Sixteen (89%) of the lingual specimens revealed infection of keratinocytes by herpes-type virus. There was no evidence of virus infection in the 12 buccal mucosal scrapings. Fungal hyphae were seen by TEM in 14 (78%) of the lingual scrapings and two (17%) of the buccal scrapings. One exfoliative specimen and two biopsy specimens were stained for Epstein-Barr virus DNA with a DNA probe. The demonstration of herpes-type virions by TEM in keratinocytes from a lesion clinically suspected to be hairy leukoplakia provides direct, objective diagnosis. Furthermore, use of exfoliative cytologic specimens provides a clinically simple, noninvasive technique.

Severe chronic EBV infection associated with specific EBV immunodeficiency and an EBNA+ T-cell lymphoma containing linear, EBV DNA.

A patient with severe chronic Epstein-Barr virus (EBV) infection (CEBVI) of 6 years duration developed an EBV+ T-cell lymphoma. To determine whether the development of the T-cell tumor was linked to EBV, we studied this patient's EBV-specific immune response and her T-cell tumor tissue for evidence of EBV infection. Peripheral blood lymphocytes from this patient were systematically studied for immune function and response to EBV. Tumor tissue was examined for EBV genome and for evidence of EBV replication. This patient failed to develop anti-EBV nuclear antigen (EBNA) antibodies and had decreased mitogen responsiveness. Her T-cells showed a broad suppression of both autologous and allogeneic B-cells, which was coincident with clinical hypoimmunoglobulinemia. A selective cytotoxic T-cell defect toward autologous EBV-infected B lymphoblasts, which could not be corrected by the addition of lymphokine-mediated T-cell help, was also documented. A lymph node biopsy taken 5 years after her clinical presentation revealed lymph node architecture completely effaced by a diffuse CD3+, CD4+, Ia+, CR2+ T-cell lymphoma containing EBNA and linear, replicating EBV DNA. Select CEBVI patients with humoral and combined cellular aberrations in the immune response to EBV may be predisposed to the development of EBV+ T-cell tumors.

Oral hairy leukoplakia in an HIV-negative renal transplant patient.

A case of oral hairy leukoplakia presenting in an HIV-negative renal transplant recipient is described. The diagnosis was confirmed by identifying Epstein-Barr viral particles in the upper prickle cell layers of the epithelium by electron microscopy and by in situ DNA hybridisation.

Epstein-Barr virus in a CD8-positive T-cell lymphoma.

In contrast to its role in B-lymphomagenesis, Epstein-Barr Virus (EBV) only incidentally has been associated with T-cell lymphomas. In the present report we describe a fourth patient with EBV-related T-cell lymphoma. The patient presented with an angio-immunoblastic lymphadenopathy (AILD)-like T-cell lymphoma. Serology was compatible with chronic Epstein-Barr (EBV) infection. After a 1-year period of waxing and waning lymphadenopathy, this lymphoma evolved to an aggressive CD8+ Immunoblastic T-cell lymphoma. A relationship with the chronic EBV infection was indicated by the finding of EBV genome in the tumor tissue by Southern blot analysis. Moreover, EBV nuclear antigen (EBNA) was detected in situ within individually defined CD8+ tumor cells by two-color immunofluorescence. Two alternative possibilities, namely that EBV primarily played a role in lymphomagenesis of the AILD-like T-cell lymphoma or that the virus was an additional oncogenic event in the final process of tumor progression to the immunoblastic lymphoma, are discussed.

Oligoclonal immunoglobulin heavy chain gene rearrangement in a childhood immunoblastic lymphoma. Presentation as a polyphenotypic atypical lymphoproliferative reaction.

The authors describe a diagnostically difficult case of childhood lymphoma that presented as an atypical polyphenotypic lymphoproliferative reaction. Initial immunophenotyping revealed the presence of IgG, IgA, kappa, and lambda within the neoplastic lymphocytes. The patient had circulating plasmacytoid lymphocytes and a polyclonal hypergammaglobulinemia. The patient died of widespread immunoblastic lymphoma in two months. Postmortem tumor DNA showed a oligoclonal pattern of immunoglobulin heavy chain gene rearrangement. Blots for T-cell receptor beta-chain rearrangement showed germline bands. Epstein-Barr virus DNA was present within tumor cells, but there was no history of prior immunosuppression or serologic evidence of Epstein-Barr virus infection. The apparent polyclonal nature of the immunoproliferation delayed the institution of chemotherapy.

[Analysis of B-lymphotropic oncogenic herpesvirus of Macaca arctoides]. / Analiz B-limfotropnogo onkogennogo virusa gerpesa makakov burykh.

Some physico-chemical and molecular-biological parameters of B-lymphotropic herpes virus of Macaca arctoides (HVMA) have been analyzed. The buoyant density of virions and nucleocapsids in a gradient of the percoll density is 1.10 and 1.14 g/ml, respectively, while that of DNA in CsCl gradient is 1.717 g/ml, which is typical for herpes viruses of primates. At the same time the homology of DNA HVMA with DNA EBV and DNA HVP is 30-45%. Differences in restrictions sites between DNA HVMA, DNA HVP and DNA EBV are determined. The intraspecific heterogeneity of HVMA strains has been detected according to restriction sites of DNA. The HVMA genome size is about 170 kb. The DNA HVMA is colinear to the EBV genome, which is also typical of B-lymphotropic herpes viruses of primates.

Regulation of c-fgr proto-oncogene expression in Burkitt's lymphoma cells: effect of interferon treatment and relationship to EBV status and c-myc mRNA levels.

The proto-oncogene c-fgr is expressed in transformed human B lymphoid cell lines and has been reported to be induced in cells infected with the Epstein-Barr virus (EBV). We have compared the levels of c-fgr mRNA in several B cell lines and have examined the effects of interferon-induced changes in growth rate of Daudi cells on the concentration of this mRNA. High levels were found in exponentially growing EBV-positive Raji and Daudi cells but the amounts in B95-8 and P3HR-1 cells (also EBV-positive) were lower than in the EBV-negative cell line Ramos. Growth inhibition of Daudi cells by interferon-alpha is preceded by a reduction in c-fgr expression, with a 56% decrease observed within 6 h. The differences in the amounts of c-fgr mRNA in the various cell lines and in control versus interferon-treated cells are similar to the differences in the c-myc mRNA contents of these cells. These results indicate that c-fgr expression bears little relationship to the EBV status of B lymphoid cell lines but may play a role, in conjunction with c-myc expression, in growth regulation by interferons. Other conditions which influence Daudi cell proliferation, such as treatment with a phorbol ester or growth to high cell density, also inhibit c-fgr expression but to a lesser extent than interferon treatment.

Epstein-Barr virus latent gene expression during the initiation of B cell immortalization.

Epstein-Barr virus (EBV) has the capacity to immortalize a subpopulation of resting B lymphocytes. Lymphoblastoid cell lines (LCL) established in this way carry the latent EBV genome as multiple copies of an extrachromosomal episome. Viral gene expression in LCLs is highly restricted; products identified correspond to a membrane protein (latent membrane protein; LMP), a nuclear antigen complex (Epstein-Barr nuclear antigens; EBNAs 1 to 6), two small RNA species (EBERs 1 and 2) and RNA species thought to encode a second membrane-associated polypeptide designated terminal protein (TP). Here we have investigated the temporal sequence of expression of the characterized 'latent' proteins during the initiation of immortalization when resting B cells are stimulated to enter and traverse the cell cycle. The analysis has been carried out on prolymphocytic leukaemia cells infected in vitro with either the immortalizing B95-8 strain of virus or the non-immortalizing P3HR1 strain. The results reveal that following B95-8 infection, a sequence of EBV expression is initiated within approximately 8 h with the synthesis of detectable levels of EBNA 2 shortly followed by EBNAs 1, 3, 4, 5 and 6. There is then a delay of approximately 40 h until the expression of LMP completes the latent pattern of proteins found in LCLs. P3HR1 infection, however, produces only transient expression of some EBNA species in a small percentage of cells after approximately 48 h. These observations suggest the failure of P3HR1 virus to immortalize may not be due solely to the absence of EBNA 2 expression and that cellular and/or virus-mediated events occur after EBNA synthesis which then facilitate efficient LMP expression and immortalization.

Characterization of N- and O-linked oligosaccharides of glycoprotein 350 from Epstein-Barr virus.

Glycoprotein 350 (gp350), the major Epstein-Barr Virus (EBV) envelope glycoprotein, has extensive N- and O-linked oligosaccharide chains. To characterize these oligosaccharide chains, [3H]glucosamine-labeled gp350 was isolated from an EBV transformed marmoset lymphoblastoid cell line (B95-8) induced to replicate EBV. Radiolabeled pronase-glycopeptides were fractionated by serial affinity chromatography and O-linked oligosaccharides released by mild alkaline borohydride treatment. Virtually all (99%) N-linked oligosaccharides were of complex type, with a predominance of tri-tetraantennary versus diantennary chains. A significant portion (28%, in term of radioactivity) of the tri-tetraantennary chains bound to leucoagglutinin-agarose, indicating an additional branch in beta(1-6)-linkage to the trimannosyl core. N-linked oligosaccharides with such a branching pattern have not been previously described in any herpesvirus glycoprotein, but have been associated with neoplastic transformation. Half of [3H]glucosamine incorporated into gp350 was recovered in O-linked oligosaccharides. The smallest chains have a core beta Gal-GalNAc disaccharide structure. Most O-linked chains have two to three N-acetylglucosamine and one N-acetylgalactosamine residues, besides the N-acetylgalactosamine residue located at the terminal reducing end, suggesting a di- or tri- N-acetyllactosamine structure. Consistent with such a structure, the size of these chains, after sialic acid removal, was that of an heptasaccharide or larger.

Oral lymphomas in HIV-infected patients: association with Epstein-Barr virus DNA.

Nine instances of oral non-Hodgkin's lymphoma occurring in homosexual male patients infected with the human immunodeficiency virus were evaluated for clinical features, histopathologic features, lymphocyte phenotypic markers, and Epstein-Barr virus (EBV) DNA. Histologically, the tumors represented immunoblastic sarcoma and small noncleaved cell lymphomas, some manifesting Burkitt-like features. Five cases exhibited positive staining with monoclonal antibody L26, a B-cell marker; none of the tumors showed evidence of a T-cell lineage with the use of monoclonal antibody UCHL 1. DNA in situ hybridization studies disclosed the presence of EBV DNA sequences in seven instances. These findings indicate that most oral lymphomas among patients with AIDS, similar to extraoral lymphomas in this population, are of B-cell lineage and harbor EBV DNA.

Distinctive charge configurations in proteins of the Epstein-Barr virus and possible functions.

The protein products of several open reading frames (ORFs) of the Epstein-Barr virus (EBV) are remarkable in their distribution of charged residues. The nuclear antigen proteins EBNA1-EBNA4 of the EBV latent state contain separate significant clusters of charge of each sign. They (excepting EBNA4) also feature distinctive periodic charge patterns [e.g., (+, O)8, (O, -, -)7] and significant tandem repeats. None of the other ORFs (about 80) of the genome possess the conjunction of these properties. Only the protein encoded from BMLF1, the first immediate early transactivator protein, contains significant multiple charge clusters and periodic charge patterns. All proteins that contain significant repeats also contain at least one significant charge cluster of a single sign. These include EBNA5 and LYDMA produced during latency and BZLF1, whose expression terminates latency and initiates productive growth. It is reasonable to conclude that these aggregate significant charge configurations and repeats are important functionally for the latent existence and for the initiation of the lytic cycle and may be characteristic of these conditions. We discuss how large multimeric protein structures bound together by clusters of unlike charge may provide a mechanism for regulation of the expression of these proteins.