The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.     

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.     

A novel membrane protein is a mouse mammary tumor virus receptor

Mouse mammary tumor virus (MMTV) infects a number of different cell types, including mammary gland and lymphoid cells, in vivo. To identify the cellular receptor for this virus, a mouse cDNA expression library was transfected into Cos-7 monkey kidney cells, and those transfected cells able to bind virus were selected by using antibody against the virus's cell surface envelope protein, gp52. One clone isolated from a library prepared from newborn thymus RNA, called MTVR, was able to confer virus binding to both monkey and human cells; this binding was blocked by anti-MTVR antibody. Moreover, transfection of MTVR into CV1 cells rendered them susceptible to infection by a murine leukemia virus-based retrovirus vector pseudotyped with the MMTV envelope protein. An epitope-tagged MTVR cofractionated with cellular membranes. Coimmunoprecipitation of the MMTV envelope protein and a MTVR-rabbit Fc fusion protein showed that these two proteins bound to each other. The MTVR sequence clone is unique, shows no homology to known membrane proteins, and is transcribed in many tissues.     

Epstein-Barr virus strain type and latent membrane protein 1 gene deletions in lymphomas in patients with rheumatic diseases

We have previously shown that long-term angiotensin-converting enzyme (ACE) inhibition prevents the increase in aortic collagen in spontaneously hypertensive rats (SHRs), independent of blood pressure reduction. More recently, we reported that the effects of ACE inhibition in the prevention of aortic collagen accumulation were related to the inhibition of angiotensin II actions on angiotensin II type 1 receptors. Aldosterone, the synthesis of which is mainly modulated by angiotensin II through type 1 receptor stimulation, is known to promote cardiac fibrosis in different experimental models. The aim of the present study was to determine whether inhibition of aldosterone formation was able to prevent aortic fibrosis in SHRs. For this purpose, we compared the effects of a 4-month treatment with the aldosterone antagonist spironolactone with the ACE inhibitor quinapril in 4-week-old SHRs. Control SHRs and Wistar-Kyoto (WKY) rats received placebo for the same period of time. At the end of treatment, in conscious SHRs vs WKY controls, quinapril completely prevented the development of hypertension, whereas spironolactone produced only a slight but significant reduction in blood pressure. Aortic hypertrophy was significantly prevented by ACE inhibition but not by spironolactone. On the contrary, aortic collagen accumulation was completely prevented by both quinapril and spironolactone. In the latter case, collagen density was significantly below that of WKY controls. These results show that in SHRs, spironolactone can markedly prevent aortic fibrosis in the presence of a very slight antihypertensive effect. It is suggested that ACE inhibition or type 1 receptor antagonist-induced prevention of aortic collagen accumulation is at least partially related to aldosterone inhibition.     

Epstein-Barr virus latent membrane protein-1 oncogene deletion in post-transplantation lymphoproliferative disorders

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a multifunctional oncoprotein. A 30-bp deletion of the 3' end of the LMP1 gene (del-LMP1) has been identified in some EBV isolates. This deleted LMP1 gene encodes a protein, altered on the carboxy terminus, which is thought to have greater oncogenic potential than the wild type. Recently, it was suggested that del-LMP1 plays a role in the development of malignant lymphomas occurring in immunocompromised patients. To further elucidate the role of del-LMP1 in post-transplantation lymphoproliferative disorders (PT-LPDs) we analyzed 58 PT-LPD lesions from 36 heart and kidney organ transplant recipients. Overall, del-LMP1 was detected in 44% of the cases. Four plasmacytic hyperplasias (36%), eight polymorphic B-cell hyperplasias/polymorphic B-cell lymphomas (38%), and five malignant lymphomas/multiple myelomas (71%) exhibited del-LMP1. Two of the three patients displaying disease progression showed wild-type LMP1 gene (w-LMP1) and one showed del-LMP1. LMP1 status remained the same in all three patients during disease progression. In patients undergoing biopsy of multiple separate PT-LPD lesions representing different clonal lymphoid proliferations, LMP1 status was the same in all of the lesions in each patient. Furthermore, although the polyclonal lesions harbor multiple EBV infectious events, they either showed w- or del-LMP1 but not both. Analysis of the tissues without an apparent PT-LPD (peripheral blood, bone marrow, or colon) revealed EBV and LMP1 type identical to that found in the lesions. In conclusion, the presence or absence of del-LMP1 in PT-LPDs does not correlate with the histopathological category or the malignant nature of the lymphoid proliferation. LMP1 status does not change during disease progression and is the same within multiple lesions occurring in the same patient regardless of their clonal relationship. These findings suggest that 1) EBV infection in patients with PT-LPDs occurs with a w- or del-LMP1-type EBV isolate and does not change once a patient acquires the virus and 2) the infection is an early event in the development of PT-LPDs and transformation is induced regardless of the type of LMP1.     

Crystal structure of Epstein-Barr virus protein BCRF1, a homolog of cellular interleukin-10

The crystal structure of Epstein-Barr virus protein BCRF1, an analog of cellular interleukin-10 (IL-10), has been determined at the resolution of 1.9 A and refined to an R-factor 0.191. The structure of this cytokine is similar to that of human IL-10 (hIL-10), forming an intercalated dimer of two 17 kDa polypeptides related by a crystallographic 2-fold symmetry axis. BCRF1 exhibits novel conformations of the N-terminal coil and of the loop between helices A and B compared to hIL-10. These regions are likely to be involved in binding of one or more components of the IL-10 receptor system, and thus the structural differences may account for the lower binding affinity and limited spectrum of biological activities of viral IL-10, compared to hIL-10.     

Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

Protein engineering of a novel constitutively active hormone-receptor complex

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha and a beta subunit that stimulates intracellular levels of cAMP via a G protein-coupled receptor. Herein we report the engineering and characterization of a novel molecule in which the receptor and its heterodimeric ligand were covalently linked in a single polypeptide chain. The hormone-receptor complex was expressed in cells transfected with this construct, but the cells were unable to bind significant amounts of exogenous hCG. However, cleavage of the hormone with a site-specific protease rendered the receptor accessible to exogenously added hormone. Cells transfected with the hCG-receptor construct contained elevated basal levels of cAMP; moreover, addition of hormone had no significant effect. These results are consistent with a strong and stable interaction between the single-chain hormone and its covalently linked receptor that results in a constitutively active complex.     

The expression of constitutively active isotypes of protein kinase C to investigate preconditioning

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta-galactosidase (beta-gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta-gal and either constitutively active mutants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta, rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p 相似文献   

Tyrosine 112 of latent membrane protein 2A is essential for protein tyrosine kinase loading and regulation of Epstein-Barr virus latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

Carboxy terminal variants of Epstein-Barr virus-encoded latent membrane protein 1 during long-term human immunodeficiency virus infection: reliable markers for individual strain identification

To assess the frequency and molecular polymorphism of malignancy-associated latent membrane protein 1 (LMP1) variants in human immunodeficiency virus type 1 (HIV-1) infection, 94 B-lymphoblastoid cell lines spontaneously derived from peripheral blood mononuclear cells (PBMC) and 30 PBMC samples at seroconversion and later (mean, 55 months) were analyzed by longitudinal comparative sequence analysis in 8 patients progressing to non-Hodgkin's lymphoma (AIDS-NHL), 7 patients to opportunistic infections, and 2 patients with long-term asymptomatic HIV-1 infection. The sequence polymorphism in the C-terminus of LMP1 was characteristic for strains harbored by individual patients, with high fidelity for strain identification. In 14 of the 17 patients, two different but characteristic LMP1 variants were identified. At HIV seroconversion in 8 of 15 patients, a 30-bp deletion (LMP1Delta) was present. Though serial analysis revealed a shift to LMP1Delta in some individuals, statistical analysis of the cohort does not support the hypothesis that accumulation of LMP1Delta variants in PBMC accounts for their observed high incidence in AIDS-NHL.     

Anti-interleukin-10 antibodies in patients with chronic active Epstein-Barr virus infection

The Epstein-Barr virus (EBV) genome encodes a protein in its BamHI C restriction fragment rightward open-reading frame-1 (designated BCRF1 or viral interleukin-10 [vIL-10]) that shares protein homology and biologic properties with human IL-10. Several EBV disorders are characterized by prolonged active EBV infection. Because continued EBV replication could allow for increased vIL-10, ELISA and immunoprecipitation were used to determine whether vIL-10 expression during chronic active EBV infection resulted in vIL-10 and IL-10 antibodies. IL-10 antibodies were assayed in patients diagnosed with chronic and acute infectious mononucleosis (CIM, AIM), nasopharyngeal carcinoma (NPC), and EBV-associated lymphoproliferative disease (LPD), as well as from healthy organ transplant patients and EBV-negative or EBV-positive persons. Whether anti-IL-10 antibodies could inhibit IL-10 biologic activity was determined. vIL-10 antibodies were found in CIM, NPC, and LPD patients and antibodies reactive to IL-10 were found in CIM patients. One CIM patient had IL-10 antibodies that neutralized IL-10 bioactivity in vitro.     

Calcification in the basal ganglia with chronic active Epstein-Barr virus infection

The objective of this study was to determine whether measurements of human CRH in the inferior petrosal sinuses could distinguish patients with Cushing's syndrome from those with pseudo-Cushing states or normal physiology. Twenty-five patients with Cushing's disease, 17 patients with the syndrome of ectopic ACTH, 7 patients with Cushing's syndrome of adrenal origin, 6 patients with pseudo-Cushing states, and 11 volunteers believed to have normal hypothalamic-pituitary-adrenal axes were studied. Basal plasma human CRH and ACTH were measured at two time points in the petrosal sinuses and in a peripheral vein. Most subjects were studied after the administration of intravenous diazepam or midazolam and fentanyl, but because of the known inhibitory effects of such sedation on CRH secretion, 2 normal volunteers and 3 patients with pseudo-Cushing states were studied without sedation. Human CRH levels were near or below the detection limit of the assay in all subjects. Although the normal volunteers and patients with pseudo-Cushing states who were studied without sedation had significantly greater inferior petrosal sinus ACTH levels than those who received sedation, there were no differences in measured human CRH levels for any of the groups. We conclude that inferior petrosal sinus human CRH levels are not easily measured in the inferior petrosal sinuses and cannot be used to determine whether individual patients may have hypersecretion of CRH causing their ACTH secretion.