Autoactivation of the Epstein-Barr virus oncogenic protein LMP1 during type II latency through opposite roles of the NF-kappaB and JNK signaling pathways

Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-kappaB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV.     

Mechanisms of EBV-mediated oncogenesis

Epstein-Barr virus (EBV) is the DNA tumor virus, which is known to be relevant to various cancers. EBV maintains latent infection in cancer cells, and there are three types of latent infection (type I-III) according to the patterns of viral latent genes expression. EBV has the ability to transform B cells into immortalized lymphoblastoid cell lines (LCL) showing type III latency, in which all latent genes are expressed. The mechanism of B-cell transformation has provided a model of EBV-associated lymphomas in immunosuppressed individuals. In type I and II latency, the limited numbers of latent genes are expressed. Previous studies have demonstrated the oncogenic functions of latent EBV genes including nuclear antigen EBNA1, membrane protein LMP1 and LMP2A. In addition, we have demonstrated that EBV-encoded small RNA EBERs play a significant role in oncogenesis. Here we summarize recent progresses in the studies on molecular mechanisms of EBV-mediated oncogenesis.     

Epstein-Barr Virus Latent Membrane Protein 2A Contributes to Anoikis Resistance through ERK Activation

Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.     

Cyclophilin A binds to AKT1 and facilitates the tumorigenicity of Epstein-Barr virus by mediating the activation of AKT/mTOR/NF-κB positive feedback loop

The AKT/mTOR and NF-κB signalings are crucial pathways activated in cancers including nasopharyngeal carcinoma (NPC), which is prevalent in southern China and closely related to Epstein-Barr virus (EBV) infection. How these master pathways are persistently activated in EBV-associated NPC remains to be investigated. Here we demonstrated that EBV-encoded latent membrane protein 1 (LMP1) promoted cyclophilin A (CYPA) expression through the activation of NF-κB. The depletion of CYPA suppressed cell proliferation and facilitated apoptosis. CYPA was able to bind to AKT1, thus activating AKT/mTOR/NF-κB signaling cascade. Moreover, the use of mTOR inhibitor, rapamycin, subverted the activation of the positive feedback loop, NF-κB/CYPA/AKT/mTOR. It is reasonable that LMP1 expression derived from initial viral infection is enough to assure the constant potentiation of AKT/mTOR and NF-κB signalings. This may partly explain the fact that EBV serves as a tumor-promoting factor with minimal expression of the viral oncoprotein LMP1 in malignancies. Our findings provide new insight into the understanding of causative role of EBV in tumorigenicity during latent infection.     

Roles of the ITAM and PY motifs of Epstein-Barr virus latent membrane protein 2A in the inhibition of epithelial cell differentiation and activation of {beta}-catenin signaling

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is important for maintenance of latency in infected B lymphocytes. Through its immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs, LMP2A is able to block B-cell receptor (BCR) signaling, bind BCR-associated kinases, and manipulate the turnover of itself and these kinases via a PY-mediated interaction with the Nedd4 family of ubiquitin ligases. In epithelial cells, LMP2A has been shown to activate the phosphatidylinositol 3'-OH kinase/Akt and beta-catenin signaling pathways. In the present study, the biological consequences of LMP2A expression in the normal human foreskin keratinocyte (HFK) cell line were investigated and the importance of the ITAM and PY motifs for LMP2A signaling effects in HFK cells was ascertained. The ITAM was essential for the activation of Akt by LMP2A in HFK cells, while both the ITAM and PY motifs contributed to LMP2A-mediated accumulation and nuclear translocation of the oncoprotein beta-catenin. LMP2A inhibited induction of differentiation in an assay conducted with semisolid methylcellulose medium, and the PY motifs were critical for this inhibition. LMP2A is expressed in the EBV-associated epithelial malignancies nasopharyngeal carcinoma and gastric carcinoma, and these data indicate that LMP2A affects cellular processes that likely contribute to carcinogenesis.     

Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma

Viral proteins expressed by EBV-associated tumors provide target Ags for immunotherapy. Adoptive T cell therapy has proven effective for posttransplant EBV-associated lymphoma in which all EBV latent Ags are expressed (type III latency). Application of immunotherapeutic strategies to tumors such as nasopharyngeal carcinoma and Hodgkin's lymphoma that have a restricted pattern of EBV Ag expression (type II latency) is under investigation. Potential EBV Ag targets for T cell therapy expressed by these tumors include latent membrane proteins (LMP) 1 and 2. A broad panel of epitopes must be identified from these target Ags to optimize vaccination strategies and facilitate monitoring of tumor-specific T cell populations after immunotherapeutic interventions. To date, LMP2 epitopes have been identified for only a limited number of HLA alleles. Using a peptide library spanning the entire LMP2 sequence, 25 CTL lines from patients with EBV-positive malignancies expressing type II latency were screened for the presence of LMP2-specific T cell populations. In 21 of 25 lines, T cell responses against one to five LMP2 epitopes were identified. These included responses to previously described epitopes as well as to newly identified HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-, B*3501-, B53-, and HLA-DR-restricted epitopes. Seven of the nine newly identified epitopes were antigenically conserved among virus isolates from nasopharyngeal carcinoma tumors. These new LMP2 epitopes broaden the diversity of HLA alleles with available epitopes, and, in particular, those epitopes conserved between EBV strains provide valuable tools for immunotherapy and immune monitoring.     

EB病毒感染下调胃癌细胞系NU-GC-3肿瘤坏死因子α的表达

To explore the mechanisms of EBV associated gastric carcinoma, TNF α expression was determined by Northern blot in EBV infected gastric carcinoma cell line NU GC 3 and the significant decrease was found (P<0.01). Furthermore, the determinations were done in the cells transfected by 4 kinds of EBV latency I genes which were expressed in EBV latently infected gastric epithelial cells. It was found that the downregulations were associated with EBV determined nuclear antigen l (EBNA1) gene and EBV encoded small RNAs (EBERs) gene (p<0.01 and p<0.05), but not with BamH I A rightward frame 0 (BARF0) gene and latent membrane protein 2A (LMP2A) gene. EBNA1 and EBERs are known to be correlated with the viral tumorigenicity, while BARF0 and LMP2A are not. TNF α downregulation caused by all the factors related to EBV tumorigenicity suggests that it may be an important mediate step in the signal transduction pathway of gastric epithelial cell canceration.     

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.     

A review of the cytomorphology of Epstein-Barr virus-associated malignancies

The Epstein-Barr virus (EBV) is a member of the herpes family of viruses and is very common in humans. EBV is most often associated with infectious mononucleosis. However, it is estimated that 1% of tumors including lymphoproliferative, epithelial and mesenchymal are linked to EBV infection. EBV has a tropism for certain epithelial cells, lymphocytes and myocytes. Like other herpesviruses, EBV has both lytic and latent phases of infection. In the latent form, EBV-encoded genes ensure the survival of the viral genome, allowing it to circumvent the host's immune surveillance by limited expression of viral proteins and carries with it the risk of neoplastic transformation. Cytologists are likely to encounter EBV-associated malignancies in cytology material but unlike other herpesviruses, EBV does not evoke a viral cytopathic effect. The manifestation of EBV-related tumors is also often variable depending upon the patient's immune status. Therefore, knowledge of the patient's EBV status and immune competence (e.g. HIV-infection or transplant-related immunosuppression) combined with the cytomorphology and results of ancillary studies are often all required to make a diagnosis of EBV-associated malignancy. This review discusses the unique cytomorphology and ancillary studies required to diagnose EBV-related neoplasms.     

Epstein-Barr virus latent membrane protein 2A mediates transformation through constitutive activation of the Ras/PI3-K/Akt Pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.     

Epstein-Barr virus infection: basis of malignancy and potential for therapy

The Epstein-Barr virus (EBV) is a human herpesvirus that is usually carried lifelong as an asymptomatic infection. EBV is the causative agent of infectious mononucleosis and has been linked to the development of several malignant tumours, including B-cell neoplasms such as Burkitt's lymphoma and Hodgkin's disease, certain forms of T-cell lymphoma, and some epithelial tumours, such as undifferentiated nasopharyngeal carcinoma and a proportion of gastric cancers. All these tumours are characterised by the presence of multiple extrachromosomal copies of the circular viral genome in the tumour cells and the expression of EBV-encoded latent genes, which appear to contribute to the malignant phenotype. An increasing understanding of the function of EBV latent genes and of the nature of the immune response to the virus is providing exciting new possibilities for the treatment of EBV-associated malignancies. For example, adoptive transfer of virus-specific cytotoxic T lymphocytes has already been of value in the treatment of EBV-positive B-cell lymphomas arising in post-transplant patients, and this approach is currently being investigated in other EBV-associated tumours. In addition, gene therapy offers the opportunity to deliver agents that might directly interfere with the function of specific EBV genes. This review summarises the role of EBV in malignancy. In particular, it focuses on the latent proteins as a basis for understanding how EBV might contribute to the process of transformation. Strategies to target EBV in tumours, potentially providing alternative therapeutic approaches, are also discussed.     

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.     

Epstein-Barr virus LMP2A alters in vivo and in vitro models of B-cell anergy, but not deletion, in response to autoantigen

A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-kappaB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-kappaB nuclear translocation independent of BCR cross-linking. Since NF-kappaB is required to bypass tolerance induction, this LMP2A-dependent NF-kappaB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.     

Sequence variations of latent membrane protein 2A in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China

Latent membrane protein 2A (LMP2A), expressed in most Epstein-Barr virus (EBV)-associated malignancies, has been demonstrated to be responsible for the maintenance of latent infection and epithelial cell transformation. Besides, it could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence variations of LMP2A in EBV-associated gastric carcinoma (EBVaGC) and healthy EBV carriers from Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were investigated. Widespread sequence variations in the LMP2A gene were found, with no sequence identical to the B95.8 prototype. No consistent mutation was detected in all isolates. The immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs in the amino terminus of LMP2A were strictly conserved, suggesting their important roles in virus infection; while 8 of the 17 identified CTL epitopes in the transmembrane region of LMP2A were affected by at least one point mutation, which may implicate that the effect of LMP2A polymorphisms should be considered when LMP2A-targeted immunotherapy is conducted. The polymorphisms of LMP2A in EBVaGC in gastric remnant carcinoma (GRC) were for the first time investigated in the world. The LMP2A sequence variations in EBVaGC in GRC were somewhat different from those in EBVaGC in conventional gastric carcinoma. The sequence variations of LMP2A in EBVaGC were similar to those in throat washing of healthy EBV carriers, indicating that these variations are due to geographic-associated polymorphisms rather than EBVaGC-associated mutations. This, to our best knowledge, is the first detailed investigation of LMP2A polymorphisms in EBVaGC in Guangzhou, southern China, where NPC is endemic.     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-α) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.