An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.     

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.     

Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.     

Latent membrane protein 2B regulates susceptibility to induction of lytic Epstein-Barr virus infection

The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.     

C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal

The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.     

Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.

The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2.     

Expression and characterization of N-terminal domain of Epstein-Barr virus latent membrane protein 2A in Escherichia coli

Latency of Epstein-Barr virus (EBV) is maintained by the transmembrane protein latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. LMP2A contains a cytoplasmic N-terminal domain composed of 119 amino acids, which provides signals that are responsible for the association with various signal molecules, resulting in negative regulation of B-cell signaling and the EBV lytic cycle. In the present study, to obtain N-terminal domain of LMP2A (LMP2A NTD, 13 kDa) in Escherichia coli for structural analysis, a strategy for obtaining the unfused form of LMP2A NTD without any fusion partners was proposed. Recombinant LMP2A NTD has previously been expressed using the GST fusion system in E. coli [Virology 268 (2000) 178, J. Virol. 71 (1997) 4752, Mol. Cell. Biol. 20 (2000) 8526]. However, we were unable to obtain untagged LMP2A NTD from this construct because of rapid proteolysis by thrombin. To overcome the proteolysis by thrombin, C-terminal His-tagged LMP2A NTD and intein-fused LMP2A NTD were prepared. As a result, LMP2A NTD without a fusion partner could be successfully obtained using non-enzymatic cleavage. The secondary structure of the recombinant LMP2A NTD was analyzed using circular dichroism. In aqueous solution, LMP2A NTD adopts an unordered structure, which was not affected by varying pH and salt concentration. In addition, any secondary structural components of LMP2A NTD were not induced in the membrane-mimicking environments, suggesting that LMP2A NTD may intrinsically have a random coil-like structure. The biological activity of recombinant LMP2A NTD was monitored by chemical shift perturbation in HSQC spectra of LMP2A NTD with or without WW domains, which result supports that the structural change induced by WW domains is restricted within narrow region.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

Syk tyrosine kinase mediates Epstein-Barr virus latent membrane protein 2A-induced cell migration in epithelial cells

Although spleen tyrosine kinase (Syk) is known to be important in hematopoietic cell development, the roles of Syk in epithelial cells have not been well studied. Limited data suggest that Syk plays alternate roles in carcinogenesis under different circumstances. In breast cancer, Syk has been suggested to be a tumor suppressor. In contrast, Syk is essential for murine mammary tumor virus-mediated transformation. However, the roles of Syk in tumor migration are still largely unknown. Nasopharyngeal carcinoma, an unusually highly metastatic tumor, expresses Epstein-Barr virus LMP2A (latent membrane protein 2A) in most clinical specimens. Previously, we demonstrated LMP2A triggers epithelial cell migration. LMP2A contains an immunoreceptor tyrosine-based activation motif, which is important for Syk kinase activation in B cells. In this study, we explored whether Syk is important for LMP2A-mediated epithelial cell migration. We demonstrate that LMP2A expression can activate endogenous Syk activity. The activation requires the tyrosine residues in LMP2A ITAM but not YEEA motif, which is important for Syk activation by Lyn in B cells. LMP2A interacts with Syk as demonstrated by coimmunoprecipitation and confocal microscopy. Furthermore, LMP2A-induced cell migration is inhibited by a Syk inhibitor and short interfering RNA. Tyrosines 74 and 85 in the LMP2A immunoreceptor tyrosine-based activation motif are essential for both Syk activation and LMP2A-mediated cell migration, indicating the involvement of Syk in LMP2A-triggered cell migration. The LMP2A-Syk pathway may provide suitable drug targets for treatment of nasopharyngeal carcinoma.     

Latent membrane protein 1 of Epstein-Barr virus stimulates processing of NF-kappa B2 p100 to p52

Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-kappa B activation pathway that depends upon the inducible processing of NF-kappa B2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of Epstein-Barr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52.RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-kappa B-signaling pathway that triggers phosphorylation and degradation of I kappa B alpha. Interestingly, LMP1 activation of these two NF-kappa B pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of I kappa B alpha phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of I kappa B alpha phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function.     

Epstein-Barr virus latent membrane protein 2A regulates c-Jun protein through extracellular signal-regulated kinase

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in both EBV-infected cells and EBV-associated malignancies. However, the function of LMP2A is still veiled. In this study, LMP2A was found to induce the kinase activities of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK. Furthermore, the downstream effector c-Jun showed hyperphosphorylation under LMP2A expression. The phosphorylation could be inhibited by the ERK pathway inhibitor PD98059, indicating that ERK may contribute to the phosphorylation of c-Jun in LMP2A-expressing cells. The impact on c-Jun phosphorylation by mitogen-activated protein kinase (MAPK) is suggested to increase c-Jun protein stability, and this was also observed in LMP2A-expressing cells by a protein synthesis inhibition assay. Moreover, LMP2A-induced cell invasion was inhibited in the presence of the ERK pathway inhibitor. Taken together, we suggest that LMP2A may exploit MAPK kinases and affect both the phosphorylation and stability of c-Jun protein. Additionally, LMP2A may thereby promote the mobility of the cells. In doing so, it may enhance the mobility of EBV-infected cells and contribute to the metastatic process of malignant cells. Here we demonstrated the first evidence of LMP2A-induced migration and the underlying pathways accounting for it.     

Latent membrane protein 1 is dispensable for Epstein-Barr virus replication in human embryonic kidney 293 cells

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.     

An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.