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  • 1
    Abstract: The effect of inhibitors of RNA (actinomycin D, Act. D) and protein synthesis (cycloheximide, CH) on induction of Epstein-Barr virus (EBV) antigens by the tumour promoter TPA and by EBV superinfection has been analysed. The data indicate that (i) concomitant treatment of cells with Act. D and TPA leads to complete suppression of virus antigen induction. Subsequent treatment of the cells with Act. D after prior exposure to TPA results in some virus antigen induction, the amount depending on the time of TPA treatment. (ii) Simultaneous treatment of the cells with TPA and CH blocks antigen expression completely. Removal of the inhibitor results in antigen expression at a comparable rate to that of CH-untreated cells. (iii) If Ch treatment is followed by addition of Act. D, virus antigen induction by TPA is completely inhibited. In contrast, superinfection of the cells with P3HR-1 EBV in the presence of CH for the same period followed by removal of the inhibitor and addition of Act. D leads to virus antigen expression by 3 h after Act. D addition. (iv) Concomitant treatment with CH and TPA followed by addition of either iododeoxyuridine or n-butyric acid results in 'superinduction'. Virtually all cells exhibit EBV-specified antigens. This implies that induction of virus antigens by tumour promoters requires the synthesis of a specific RNA, that this RNA increases in concentration during the induction period and that the same RNA is not required for EBV transcription after exogenous infection.
    Type of Publication: Journal article published
    PubMed ID: 6262437
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  • 2
    Keywords: CELLS ; IN-VITRO ; CELL ; Germany ; IMAGES ; QUANTIFICATION ; GENOME ; PROTEIN ; PROTEINS ; MOLECULES ; COMPLEX ; COMPLEXES ; DNA ; MOLECULE ; MATURATION ; virus ; ESCHERICHIA-COLI ; MEMBRANE ; PHENOTYPE ; REPLICATION ; ABNORMALITIES ; HERPES-SIMPLEX-VIRUS ; EPSTEIN-BARR-VIRUS ; Epstein-Barr virus ; development ; USA ; VIRAL-DNA ; GENOMES ; MESSENGER-RNA DEGRADATION ; EGRESS ; HOST SHUTOFF FUNCTION ; NUCLEASE ; STRAND EXCHANGE ; VIRAL-DNA REPLICATION
    Abstract: The Epstein-Barr virus (EBV) alkaline exonuclease BGLF5 has previously been recognized to contribute to immune evasion by downregulating production of HLA molecules during virus replication. We have constructed a BGLF5-null virus mutant to determine BGLF5's functions during EBV viral replication. Quantification of virus production in permissive 293 cells carrying a Delta BGLF5 genome identified a 17- to 21-fold reduction relative to complemented or wild-type controls. Detailed monitoring of Delta BGLF5 replication evidenced an impaired virus nucleocapsid maturation, a reduced primary egress and a 1.4-fold reduction in total viral DNA synthesis. Delta BGLF5 single-unit-length viral genomes were not only less abundant but also migrated faster than expected in gel electrophoresis. We concluded that BGLF5 pertained both to the generation and to the processing of viral linear genomes. Delta BGLF5 phenotypic traits were reminiscent of those previously identified in a mutant devoid of UL12, BGLF5' s homolog in herpes simplex virus type 1, and indeed UL12 was found to partially complement the Delta BGLF5 phenotype. However, BGLF5-specific functions could also be identified; the nuclear membrane of replicating cells displayed images of reduplication and complex folding that could be completely corrected by BGLF5 but not UL12. Similar nuclear abnormalities were previously observed in cells transfected with BFLF2 and BFRF1, two viral proteins crucial for EBV nuclear egress. Interestingly, Delta BGLF5 cells produced more BFLF2 than wild-type or complemented counterparts. The present study provides an overview of BGLF5' s functions that will guide future molecular studies. We anticipate that the 293/Delta BGLF5 cell line will be instrumental in such developments
    Type of Publication: Journal article published
    PubMed ID: 19264771
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  • 3
    ISSN: 1432-1076
    Keywords: Epstein-Barr virus ; Lymphoproliferative syndrome ; Inherited immunodeficiency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report on a 30-month-old previously healthy Turkish boy who presented with fever, hepatosplenomegaly and generalized lymphadenopathy. He died 4 months after admission in spite of treatment with steroids, acycloguanosine and cyclophosphamide. Epstein-Barr virus (EBV) DNA was detected in the patient's bone marrow and in a lymph node biopsy. Cells from the lymph node biopsy showed monoclonal rearrangements of immunoglobulin heavy chain genes but no rearrangements of T-cell receptor β-chain genes or immunoglobulin kappa chain genes. Serological data indicated chronic active EBV infection. There was a slight increase of CD8 positive cells in peripheral blood and a normal response to T-cell mitogens. However, T-cell lines established with interleukin 2 from lymph node biopsy completely failed to kill autologous EBV-transformed B-cells and K 562 target cells. Moreover, in regression tests the patient's peripheral blood mononuclear cells completely failed to limit outgrowth of autologous EBV infected B-cells. We conclude that the patient's selective immunodeficiency had led to the rapid development of EBV-associated monoclonal lymphoproliferation.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-2307
    Keywords: Key words Gonadoblastoma ; Germ cell tumour ; Human endogenous retrovirus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Gonadoblastomas are rare tumours of abnormal or dysgenetic gonads, often transforming to invasive seminomatous and nonseminomatous germ cell tumours (GCT). Because of the intimate association of noninvasive and invasive lesions, gonadoblastoma may provide clues as to the molecular pathogenesis of GCT. We studied the expression of the human endogenous retrovirus (HERV)-K gag gene in eight gonadoblastomas arising in phenotypically female patients, including two newborn girls. We also studied testicular biopsies with immature Sertoli cell nodules harbouring neoplastic germ cells, a lesion with morphological resemblance to gonadoblastoma. In five gonadoblastomas, invasive seminoma/dysgerminoma was noted, in two cases with formation of additional GCT components. HERV-K gag transcripts were found with moderate levels in gonocytes of all gonadoblastomas and in neoplastic germ cells in testicular Sertoli cell nodules. All invasive GCT except for teratomas displayed HERV-K transcripts. Thus, expression of HERV-K is induced during fetal or embryonal development and precedes invasive GCT formation. Although the specific role of HERV-K expression remains unknown, the findings place HERV-K expression in an appropriate time frame for it to have a role in the molecular pathogenesis of GCT and suggest a precursor-invasive tumour relationship for ovarian GCT equivalent to the more common carcinoma in situ of the testis and testicular GCT.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Nef protein, encoded by the regulatorynef gene of human immunodeficiency virus type 1 (HIV-1), was expressed in the B-cell line Raji. The cells were stably transfected with plasmids containing thenef transcriptional cassette. They expressed Nef with an Mr of 27,000; the yield could be augmented by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The intracellular localization of Nef was analyzed applying immunofluorescence microscopy using a confocal laser scanning microscope. The antigen was stained with a monoclonal antibody directed against the N-terminal part of Nef. The experiments revealed that in non-dividing cells Nef is present both in the cytoplasm and the nucleus while in dividing cells the viral protein is present in the cytoplasm and at the nuclear membrane.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83–93, 175–190 and 86–166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Reactivation of EBV infection is common in immunocompromised individuals. We determined specific antibodies to EBV-encoded nuclear antigens (EBNA) 1 and 2 in 102 sera of HIV-infected individuals. Anti-EBNA1/anti-EBNA2 ratio (E1/E2) is less than 1 in chronic infection and exceeds 1 in healthy EBV-positive carriers. 52% of cases had E1/E2〈1. E1/E2 decreased remarkably during the progression of HIV infection. Detectable HIV-Antigen, decline of CD4 + cell count and CD4 + /CD8 + ratio were correlated with an increasing prevalence of E1/E2 below 1. We conclude that determination of E1/E2 is useful in immunocompromised patients.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The 1.34 kb BcII-BgIII-fragment of the BamHI-M region of Epstein-Barr virus genome, comprising the complete BMRF1 open reading frame [1], was cloned into the tryptophan regulated E. coli expression vector pATH1. The resulting fusion protein, having a molecular weight of 80kd, is recognized not only by anti-early antigen (EA)-positive human sera but also by the monoclonal antibody R3 directed against the diffuse component of EA (EA-D) [29]. A possible use for this fusion protein as an indicator protein in diagnosis of IgA antibodies against EA-D is presented.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The integrase (IN) protein of HIV-1 was expressed as a processed and a non-processed protein in the eucaryotic baculovirus expression system. In immunoblots we could demonstrate that recombinant baculoviruses containing the completegag andpol reading frames of HIV-1 expressed a gag/pol precursor polyprotein. The specific proteolytic activity of the recombinant protease on the gag and pol precursor proteins was used for the generation of processed gag (p 17, p 24, p 16) and pol (RT/RNaseH, IN) proteins. The non-processed IN protein, expressed as a polyhedrin fusion protein, was produced at much higher level than the processed protein.
    Type of Medium: Electronic Resource
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