Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

In nasopharyngeal carcinoma cells, Epstein-Barr virus LMP1 interacts with galectin 9 in membrane raft elements resistant to simvastatin

Nasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.     

PY motifs of Epstein-Barr virus LMP2A regulate protein stability and phosphorylation of LMP2A-associated proteins

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.     

Cytopathic effects induced by Epstein-Barr virus replication in epithelial nasopharyngeal carcinoma hybrid cells.

NPC-KT cl.S61, a subclone derived from an epithelial-nasopharyngeal carcinoma hybrid cell line (NPC-KT), showed cytopathic changes characteristic of herpesvirus replication, including formation of multinucleated giant cells and inclusion bodies, when Epstein-Barr virus replicative cycle was induced by 5-iodo-2'-deoxyuridine. Acyclovir (an inhibitor of herpesvirus DNA polymerase), Epstein-Barr virus-immune human serum, or 2-deoxyglucose (an inhibitor of the glycosylation) interfered with syncytium formation, indicating that a virus-specified glycoprotein belonging to the late group is responsible for cell fusion induced by Epstein-Barr virus replication in cl.S61 cells.     

Potential selection of LMP1 variants in nasopharyngeal carcinoma

Seven distinct sequence variants of the Epstein-Barr virus latent membrane protein 1 (LMP1) have been identified by distinguishing amino acid changes in the carboxy-terminal domain. In this study the transmembrane domains are shown to segregate identically with the distinct carboxy-terminal amino acid sequences. Since strains of LMP1 have been shown to differ in abundance between blood and throat washes, nasopharyngeal carcinomas (NPCs) from areas of endemicity and nonendemicity with matching blood were analyzed by using a heteroduplex tracking assay to distinguish LMP1 variants. Striking differences were found between the compartments with the Ch1 strain prevalent in the NPCs from areas of endemicity and nonendemicity and the B958 strain prevalent in the blood of the endemic samples, whereas multiple strains of LMP1 were prevalent in the blood of the nonendemic samples. The possible selection against the B958 strain appearing in the tumor was highly significant (P < 0.0001). Sequence analysis of the full-length LMP1 variants revealed changes in many of the known and computer-predicted HLA-restricted epitopes with changes in key positions in multiple, potential epitopes for the specific HLA of the patients. These amino acid substitutions at key positions in the LMP1 epitopes may result in a reduced cytotoxic-T-lymphocyte response. These data indicate that strains with specific variants of LMP1 are more likely to be found in NPC. The predominance of specific LMP1 variants in NPC could reflect differences in the biologic or molecular properties of the distinct forms of LMP1 or possible immune selection.     

Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.     

A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1.

Recent cDNA cloning and sequencing of two Epstein-Barr virus (EBV)-specific mRNAs from latently infected cultures revealed that these RNAs are encoded across the fused terminal repeats of the viral genome and that they are likely to encode two nearly identical proteins with the same transmembrane domains. The smaller predicted protein (LMP2B) lacks 119 amino-terminal amino acids found in the larger one (LMP2A). To test whether these proteins are expressed in latently infected lymphocytes, antibodies to the LMP2 proteins were derived by immunizing rabbits with TrpE-LMP2A fusion proteins. Affinity-purified LMP2-specific antibodies recognized 54- and 40-kilodalton proteins, corresponding to LMP2A and LMP2B, in immunoblots of rodent fibroblasts stably transfected with eucaryotic expression plasmids containing either the LMP2A or LMP2B cDNA. Similar-size proteins were also identified in immunoblots of latently infected lymphocytes. LMP2A localized to membranes in cellular fractionation studies. In immunofluorescent studies, LMP2 localized in the plasma membrane of EBV-infected lymphocytes, with the majority of reactivity confined to the region of the LMP1 patch. This reactivity was detected in almost all lymphoblastoid cells latently infected with EBV.     

WW- and SH3-domain interactions with Epstein-Barr virus LMP2A

Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1) in nasopharyngeal epithelial cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.