LMP1, a viral relative of the TNF receptor family,signals principally from intracellular compartments

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.     

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C > LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 > LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.     

鼻咽癌病人和正常人群中EB病毒特异性T细胞对靶抗原的识别和应答

为探讨痘苗病毒表达的Eoskein-Barr病毒（EBV）核抗原1、4（EBNA1、4）和潜伏膜蛋白1、2（LMP1、2）,在不同人群的特异性T细胞杀伤9CTL）中的作用,采集EBV阴性正常人、未经治疗的鼻咽癌（NPC）病人的EBV－IgA/VCA阳性者各10人的周围血淋巴单核细胞（PBMC）,用EBV转化B淋巴细胞,建立类淋巴母细胞（LCL）,用LCL刺激自休的T淋巴细胞作为效应细胞,以LCL感染重组痘苗病毒表达的EBNA1、4和LMP1、2为靶细胞,以^51Cr释放法检测EBV特异性CTL所识别的靶抗原。结果表明,EBV－LMP1、2可能既是EBV特异性T细胞的刺激抗原,又是其识别的靶抗原。将采集的30例试验者的各5 单克隆T细胞株分别检测HLA-Ⅰ型（A、B、C）,按照不同型别寻找相对应的EBNA1、4和LMP1、2的不同合成肽,应用酶免疫吸附斑点法（Elispot）检测EBV特异性CD8^ 的CTL应答。结果显示：10例正常人中9人有特异的LMP2应答,4人有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的LMP2,2人有特异的EBNA1,3个有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的EBNA1,3人有特异的EBNA4应答,在10例EBV－IgA/VCAbj ntg k ,6人有特异的LMP2,5人有特异的EBNA4应答。所有的试验者均未发现LMP1的特异性应答。     

Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter

The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle.     

Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.     

Biological differences between epstein-barr virus (EBV) strains with regard to lymphocyte transforming ability, superinfection and antigen induction

EB virus (EBV) preparations derived from various producing lymphoblastoid cell lines (LCL) differed in their biological properties, as judged by the following four tests: (1) cord blood lymphocyte (CBL) transformation into EBV-carrying LCL; (2) early antigen (EA) induction in Raji cells; (3) inhibition of Raji cell growth; (4) induction of the EBV-determined nuclear antigen (EBNA) in CBL. B95-8 virus transformed and induced EBNA in CBL but did not induce EA in Raji cells, nor did it inhibit their growth. P3HR-1 virus did not transform CBL, induced no EBNA or EA in CBL, but induced EA in Raji cells and inhibited their growth. EBV isolated from the QIMR-WIL, 833L, F137 and cb-8-7 LCL resembled the B95-8 virus with regard to its biological activity (CBL transformation, EA induction in and growth inhibition of Raji cells). Transformation of CBL as contrasted to EA induction in, and growth inhibition of Raji cells thus appear as mutually exclusive viral functions.     

Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma

Virus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4(+) T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8(+) T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8(+) memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4(+) memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4(+) and CD8(+) T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.     

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.     

Virus and cell RNAs expressed during Epstein-Barr virus replication

Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was approximately 5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol. 74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-kappaB responsive genes including IkappaBalpha and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication.     

Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors

Epstein-Barr virus (EBV) latency gene expression in lymphoblastoid cell lines is regulated by EBNA2. However, the factors regulating viral expression in EBV-associated tumors that do not express EBNA2 are poorly understood. In EBV-associated tumors, EBNA1 and frequently LMP1 are synthesized. We found that an alternative latent membrane protein 1 (LMP1) promoter, L1-TR, located within the terminal repeats is active in both nasopharyngeal carcinoma and Hodgkin's disease tissues. Examination of the L1-TR and the standard ED-L1 LMP1 promoters in electrophoretic mobility shift assays revealed that both promoters contain functional STAT binding sites. Further, both LMP1 promoters responded in reporter assays to activation of JAK-STAT signaling. Cotransfection of JAK1 or v-Src or treatment of cells with the cytokine interleukin-6 upregulated expression from ED-L1 and L1-TR reporter plasmids. Cotransfection of a dominant negative STAT3 beta revealed that STAT3 is likely to be the biologically relevant STAT for EBNA1 Qp and LMP1 L1-TR promoter regulation. In contrast, LMP1 expression from ED-L1 was not abrogated by STAT3 beta, indicating that the two LMP1 promoters are regulated by different STAT family members. Taken together with the previous demonstration of JAK-STAT activation of Qp driven EBNA1 expression, this places two of the EBV genes most commonly expressed in tumors under the control of the same signal transduction pathway. Immunohistochemical analyses of nasopharyngeal carcinoma tumors revealed that STAT3, STAT5, and STAT1 are constitutively activated in these tumors while STAT3 is constitutively activated in the malignant cells of Hodgkin's disease. We hypothesize that chronic or aberrant STAT activation may be both a necessary and predisposing event for EBV-driven tumorigenesis in immunocompetent individuals.     

Epstein-Barr virus nuclear antigen 2 trans-activates the cellular antiapoptotic bfl-1 gene by a CBF1/RBPJ kappa-dependent pathway

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-kappaB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-J kappa), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-kappaB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.     

Epstein-barr virus (EBV) nuclear protein 2-induced disruption of EBV latency in the Burkitt's lymphoma cell line Akata: analysis by tetracycline-regulated expression

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.