Evolution of Two Types of Rhesus Lymphocryptovirus Similar to Type 1 and Type 2 Epstein-Barr Virus

Rhesus monkeys and other nonhuman Old World primates are naturally infected with lymphocryptoviruses (LCV) that are closely related to Epstein-Barr virus (EBV). A rhesus LCV isolate (208-95) was derived from a B-cell lymphoma in a simian immunodeficiency virus-infected rhesus macaque. The EBNA-2 homologues from 208-95 and a previous rhesus LCV isolate (LCL8664) were polymorphic on immunoblotting, so the EBNA-2 genes from these two rhesus LCV were cloned, sequenced, and compared. The EBNA-2 genes have 40% nucleotide and 41% amino acid identities, and the differences are similar to those between the type 1 and type 2 EBV EBNA-2. Sequence from a portion of the LMP1 gene which is extremely divergent among different LCV was virtually identical between the 208-95 and LCL8664 strains, confirming a common rhesus LCV background. Thus, the EBNA-2 polymorphism defines the presence of two different rhesus LCV types, and both rhesus LCV types were found to be prevalent in the rhesus monkey population at the New England Regional Primate Research Center. The existence of two rhesus LCV types suggests that the selective pressure for the evolution of two LCV types is shared by human and nonhuman primate hosts.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.     

Complete Nucleotide Sequence of the Rhesus Lymphocryptovirus: Genetic Validation for an Epstein-Barr Virus Animal Model

We sequenced the rhesus lymphocryptovirus (LCV) genome in order to determine its genetic similarity to Epstein-Barr virus (EBV). The rhesus LCV encodes a repertoire identical to that of EBV, with 80 open reading frames, including cellular interleukin-10, bcl-2, and colony-stimulating factor 1 receptor homologues and an equivalent set of viral glycoproteins. The highly conserved rhesus LCV gene repertoire provides a unique animal model for the study of EBV pathogenesis.     

Down-regulation of EBV-LMP1 radio-sensitizes nasal pharyngeal carcinoma cells via NF-κB regulated ATM expression

Background

The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice.

Results

In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells.

Conclusions

Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.     

A mutation in Epstein-Barr virus LMP2A reveals a role for phospholipase D in B-Cell antigen receptor trafficking

Epstein-Barr virus (EBV) latent infection of B cells blocks the interrelated signaling and antigen-trafficking functions of the BCR through the activity of its latent membrane protein 2A (LMP2A). At present, the molecular mechanisms by which LMP2A exerts its control of BCR functions are only poorly understood. Earlier studies showed that in B cells expressing LMP2A containing a tyrosine mutation at position 112 in its cytoplasmic domain (Y112-LMP2A), the BCR could initiate signaling but could not properly traffic antigen for processing. Here, we show that BCR signaling in Y112-LMP2A-expressing cells is attenuated with a reduction in both the degree and duration of phosphorylation of key components of the BCR signaling cascade including Syk, BLNK, PI3K, and Btk. Notably, Y112-LMP2A expression completely blocked the BCR-induced activation of phospholipase D (PLD), a lipase implicated in the intracellular trafficking of a variety of surface receptors. We show that blocking PLD activity, by expressing Y112-LMP2A, treating cells with the PLD inhibitor 1-butanol or reducing PLD expression by siRNA, blocked BCR trafficking to class II-containing compartments. Moreover, Y112-LMP2A expression blocked the recruitment of phosphorylated forms of the downstream BCR signaling components, Erk and JNK, through both PLD-dependent and PLD-independent mechanisms. Thus, the investigation of the mechanism by which Y112-LMP2A blocks BCR function revealed an essential role for PLD in BCR trafficking for antigen processing.     

Infection of Human B Lymphocytes with Lymphocryptoviruses Related to Epstein-Barr Virus

Lymphocryptoviruses (LCVs) naturally infecting Old World nonhuman primates are closely related to the human LCV, Epstein-Barr virus (EBV), and share similar genome organization and sequences, biologic properties, epidemiology, and pathogenesis. LCVs can efficiently immortalize B lymphocytes from the autologous species, but the ability of a given LCV to immortalize B cells from other Old World primate species is variable. We found that LCV from rhesus monkeys did not immortalize human B cells, and EBV did not immortalize rhesus monkey B cells. In this study, baboon LCV could not immortalize human peripheral blood B cells but could readily immortalize rhesus monkey B cells. Thus, efficient LCV-induced B-cell immortalization across distant Old World primate species appears to be restricted by a species-specific block. To further characterize this species restriction, we first cloned the rhesus monkey LCV major membrane glycoprotein and discovered that the binding epitope for the EBV receptor, CD21, was highly conserved. Stable infections of human B cells with recombinant amplicons packaged in rhesus monkey or baboon LCV envelopes were also consistent with a species-restricted block occurring after virus binding and penetration. Transient infections of human B cells with simian LCV resulted in latent LCV EBNA-2 gene expression and activation of cell CD23 gene expression. EBV-immortalized human B cells could be coinfected with baboon LCV, and the simian virus persisted and replicated in human B cells. Thus, several lines of evidence indicate that the species restriction for efficient LCV-induced B-cell immortalization occurs beyond virus binding and penetration. This has important implications for the study of LCV infection in Old World primate models and for human xenotransplantation where simian LCVs may be inadvertently introduced into humans.     

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.     

A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus

STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.     

Modification of Cepsilon mRNA expression by EBV-encoded latent membrane protein 1

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cepsilon mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cepsilon mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cepsilon mRNA levels were decreased in the splenocytes derived from the immunized rats. Cepsilon mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

Proteome analysis of the transformation potential of the Epstein–Barr virus-encoded latent membrane protein 1 in nasopharyngeal epithelial cells NP69

Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.     

Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1.

Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.