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Biomedical subjects

E Kieff

Publications and source records attributed to E Kieff.

At least 55 records · Page 3Linked to original sources
PubMed1996

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1.

Nonhuman primates are naturally infected with a B-lymphotropic herpesvirus closely related to Epstein-Barr virus (EBV). These simian EBV share considerable genetic, biologic, and epidemiologic features with human EBV, including virus-induced tumorigenesis. However, latent, transformation-associated viral genes demonstrate marked sequence divergence among species despite the conserved functions. We have cloned the latent membrane protein 1 (LMP1) homologs from the simian EBV naturally infecting baboons (cercopithicine herpesvirus 12, herpesvirus papio) and rhesus monkeys (cercopithicine herpesvirus 15) for a comparative study with the human EBV oncogene. The transmembrane domains are well conserved, but there is striking sequence divergence of the carboxy-terminal cytoplasmic domain essential for B-cell immortalization and interaction with the tumor necrosis factor receptor signaling pathway. Nevertheless, the simian EBV LMP1s retain most functions in common with EBV LMP1, including the ability to induce NF-(kappa)B activity in human cells, to bind the tumor necrosis factor-associated factor 3 (TRAF3) in vitro, and to induce expression of tumor necrosis factor-responsive genes, such as ICAM1, in human B lymphocytes. Multiple TRAF3 binding sites containing a PXQXT/S core sequence can be identified in the simian EBV LMP1s by an in vitro binding assay. A PXQXT/S-containing sequence is also present in the cytoplasmic domain of the Hodgkin's disease marker, CD30, and binds TRAF3 in vitro. The last 13 amino acids containing a PXQXT/S sequence are highly conserved in human and simian EBV LMP1 but do not bind TRAF3, suggesting a distinct role for this conserved region of LMP1. The conserved TRAF3 binding sites in LMP1 and the CD30 Hodgkin's disease marker provides further evidence that a TRAF3-mediated signal transduction pathway may be important in malignant transformation.

Amino Acid Sequence↗
PubMed1996

A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes.

We have isolated a cDNA encoding a novel hematopoietin receptor family member related to the p40 subunit of interleukin-12 and to the ciliary neurotrophic factor receptor, whose expression is induced in B lymphocytes by Epstein-Barr virus (EBV) infection. This gene, which we have designated EBV-induced gene 3 (EBI3), encodes a 34-kDa glycoprotein which lacks a membrane-anchoring motif and is secreted. Despite the absence of a membrane-anchoring motif and of cysteines likely to mediate covalent linkage to an integral membrane protein, EBI3 is also present on the plasma membrane of EBV-transformed B lymphocytes and of transfected cells. Most newly synthesized EBI3 is retained in the endoplasmic reticulum in an endoglycosidase H-sensitive form associated with the molecular chaperone calnexin and with a novel 60-kDa protein. EBI3 is expressed in vivo by scattered cells in interfollicular zones of tonsil tissue, by cells associated with sinusoids in perifollicular areas of spleen tissue, and at very high levels by placental syncytiotrophoblasts. EBI3 expression in vitro is induced in EBV-negative cell lines by expression of the EBV latent infection membrane protein-1 and in peripheral blood mononuclear cells by pokeweed mitogen stimulation. EBI3 maps to chromosome 19p13.2/3, near genes encoding the erythropoietin receptor and the cytokine receptor-associated kinase, Tyk2. EBI3 synthesis by trophoblasts and by EBV-transformed cells and similarities to interleukin-12 p40 are compatible with a role for EBI3 in regulating cell-mediated immune responses.

Amino Acid Sequence↗
PubMed1996

The N-terminal half of EBNA2, except for seven prolines, is not essential for primary B-lymphocyte growth transformation.

Previous molecular genetic analyses of Epstein-Barr virus nuclear protein 2 (EBNA2) identified a negative effect of deletion of codons 19 to 33 on transformation and gene transactivation, while deletion of codons 19 to 110 was a null mutation for transformation and gene transactivation. We here report the surprising finding that codons 2 to 88, which encode the highly conserved unique N terminus (amino acids 1 to 58) and most of the polyproline repeat (amino acids 59 to 95), can be deleted with only minimal effects on transformation. Codons 97 to 122 can also be deleted with only minimal effects on transformation. However, deletion of 35 of the 37 prolines (amino acids 59 to 93) or deletion of codons 2 to 95 results in a null transforming phenotype. Although EBNA2 from which codons 59 to 93 were deleted was a null mutation for transformation, it was similar to some transforming mutants of EBNA2 in abundance, in interaction with RBPJK, and in transactivation of the LMP1 promoter in transient transfection assays. These data indicate that between three and seven prolines are critical for EBNA2 structure or for intermolecular interaction. Aside from these seven prolines, codons encoding the rest of the N-terminal half (amino acids 2 to 230) of EBNA2 are nonessential for primary B-lymphocyte growth transformation.

Antigens, Viral↗
PubMed1996

The amino-terminal domains of Epstein-Barr virus nuclear proteins 3A, 3B, and 3C interact with RBPJ(kappa).

The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. We now show that EBNA3A and EBNA3B expressed in non-EBV-infected Burkitt tumor lymphoblasts are similar to EBNA3C in binding to glutathione S-transferase-RBPJkappa in vitro and in coimmunoprecipitating from cell lysates with antibody to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis.

Antigens, Viral↗
PubMed1996

EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein-Barr virus-transformed B lymphocytes.

Although genetic and biochemical data indicate that the cell protein RBPJkappa is a mediator of EBNA-2 and EBNA-3C effects on transcriptional regulatory elements, the extent of association of these Epstein-Barr virus nuclear proteins with RBPJkappa in transformed B lymphocytes has not been determined. We now report that most of the EBNA-2 and at least 20% of the EBNA-3C coimmunoprecipitated with RBPJkappa from extracts of transformed B lymphocytes that contained most of the cellular EBNA-2 and EBNA3C. Both proteins are associated preferentially with the smaller of the two RBPJkappa isoforms. EBNA-2-RBPJkappa complexes do not contain EBNA-3C, and EBNA-3C-RBPJkappa complexes do not contain EBNA-2. Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody. Promoters with RBPJkappa sites in their regulatory elements are likely to be differentially regulated by these RBPJkappa-EBNA-2 and RBPJkappa-EBNA-3 complexes.

Antigens, Viral↗
PubMed1996

Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation.

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.

B-Lymphocytes↗
PubMed1996

Circulating human dendritic cells differentially express high levels of a 55-kd actin-bundling protein.

This study was initiated to examine the differential expression of an evolutionary conserved human 55-kd actin-bundling (p55) protein that is induced in B lymphocytes by Epstein-Barr virus infection. Our study demonstrates that p55 is specifically expressed at constitutively high levels in human peripheral blood dendritic cells and lymph node (interdigitating) dendritic cells. Blood dendritic cells constitute a minority (< 2%) of all blood leukocytes but are a distinct population of potent antigen-presenting cells. Immunofluorescence microscopy with a monoclonal antibody specific for p55 showed that 87% of peripheral blood dendritic cells stained brightly in the cytoplasm and in the veiled cytoplasmic extensions. In contrast, monocytes, granulocytes, T cells, and B lymphocytes showed no expression of the p55 protein. Western blot analysis confirmed that only the dendritic cell component of peripheral blood expressed high levels of p55. Staining of human lymph node sections demonstrated selective expression of the p55 antigen by dendritic cells in the T-cell-dependent areas but not in the B cell follicles. p55 is likely to be involved in the organization of a specialized microfilament cytoskeleton in the dendritic cells, and the anti-p55 antibody should be useful for further characterization of this important population of antigen-presenting cells in clinical transplantation, HIV-1 pathogenesis, and autoimmune diseases.

Actins↗
PubMed1995

A novel Epstein-Barr virus glycoprotein gp150 expressed from the BDLF3 open reading frame.

Analysis of cDNAs mapping to the BamHI D fragment of the Epstein-Barr virus (EBV) genome indicates that the BDLF3 open reading frame, which is predicted to encode a type 1 membrane protein of 234 amino acids, is expressed as an unspliced message. Expression of the open reading frame as a recombinant protein in vaccinia virus reveals a glycoprotein that has both N- and O-linked sugars. Antibodies made to the recombinant protein immunoprecipitate a late glycoprotein with a mobility of approximately 150,000 Da from EBV-producing cells. The glycoprotein is associated with the virion. Antibodies to it appear to react primarily with carbohydrate and do not demonstrate neutralizing activity.

Amino Acid Sequence↗
PubMed1995

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Adenosine Triphosphatases↗
PubMed1995

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family.

The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family.

Alternative Splicing↗
PubMed1995

Integral membrane protein 2 of Epstein-Barr virus regulates reactivation from latency through dominant negative effects on protein-tyrosine kinases.

An Epstein-Barr virus-encoded protein, LMP2, blocks the effects of surface immunoglobulin (slg) cross-linking on calcium mobilization and on lytic reactivation of EBV in latently infected and growth-transformed primary human B lymphocytes. In wild-type EBV-transformed cells, LMP2 is constitutively tyrosine phosphorylated and is associated with Lyn and Syk protein-tyrosine kinases (PTKs). Baseline Lyn PTK activity is substantially reduced, and slg cross-linking fails to activate Lyn, Syk, Pl3-K, PLC gamma 2, Vav, Shc, and MAPK. Syk, Pl3-K, PLC gamma 2, and Vav are constitutively tyrosine phosphorylated, and their tyrosine phosphorylation does not change following slg cross-linking. In contrast, cross-linking slg on cells transformed by LMP2 null mutant EBV recombinants triggers the same protein tyrosine kinase cascade as in noninfected B lymphocytes. These data are consistent with a model in which LMP2 is a constitutive dominant negative modulator of slg receptor signaling through its effects on Lyn, Syk, or regulators of these kinases.

Antigens, Viral↗
PubMed1995

Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1.

Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.

Antigens, Viral↗
PubMed1995

The Epstein-Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein.

The Epstein-Barr virus nuclear antigen 2 (EBNA-2) acidic domain is essential for B-lymphocyte growth transformation and can activate transcription when brought to a promoter by a sequence-specific DNA-binding domain. We now show that the EBNA-2 acidic domain has slightly less activity than the proteotypic acidic transactivator VP16 in depleting nuclear extracts of basal transcription activity. Like VP16, EBNA-2 associates with TFIIB, TAF40, and RPA70. However, EBNA-2 has much less avidity for TATA-binding protein. A Trp-to-Thr mutation within the acidic domain abolishes EBNA-2 transactivating activity and greatly compromises the association with TFIIB, TAF40, and RPA70, establishing a genetic linkage between transactivating activity and these associations.

Antigens, Viral↗
PubMed1995

The Epstein-Barr virus nuclear antigen leader protein associates with hsp72/hsc73.

Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) is important for primary B-lymphocyte growth transformation. We now demonstrate that the W repeat-encoded domain of EBNA-LP significantly associates with proteins of the heat shock protein 70 family (hsp72/hsc73). hsp72/hsc73 may mediate the previously observed interaction between EBNA-LP and the retinoblastoma protein or p53.

Amino Acid Sequence↗
PubMed1995

The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues.

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.

Antigens, Viral↗