Encoded By Epstein-Barr Virus Research Articles

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction

ABSTRACT Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-β induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle’s balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation.

The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation.IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.

MAPK/c-Jun signaling pathway contributes to the upregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL induced by Epstein-Barr virus-encoded BARF1 in gastric carcinoma cells.

BARF1, encoded by Epstein-Barr virus (EBV), has been hypothesized to function as an oncogene, which was expressed in gastric carcinoma cells. Additionally, it has been reported that the anti-apoptotic function is closely associated with the expression of the B-cell lymphoma-2 (Bcl-2) protein. In addition, the signaling pathway has been reported to be involved in numerous diseases, including the mitogen-activated protein kinase (MAPK) cascade. In order to study the specific mechanism of anti-apoptotic function, BARF1-stably-expressing immortalized normal human embryo gastric epithelial cell line GES1 (GES-BARF1), and well-, moderately- and poorly-differentiated gastric carcinoma cell lines, MKN28 (which has been reported to be contaminated with the moderately-differentiated MKN74 gastric carcinoma cell line), SGC7901 and BGC823 (MKN-BARF1, SGC-BARF1 and BGC-BARF1, respectively) (GCC-BARF1) were constructed, with transfection of cells with the empty vector pSG5 acting as controls. Western blot analysis was performed to analyze the protein expression and the phosphorylation levels. Compared with the controls, it was found that the protein expression levels of c-Jun, Bcl-2 and B-cell lymphoma-extra large (Bcl-xL), as well as the phosphorylation levels of c-Jun, c-Jun N-terminal kinase (JNK) 1/2/3, p38 and extracellular signal-regulated kinase (ERK) 1/2 proteins were upregulated in 3 GCC-BARF1 but not significantly changed in GES-BARF1. The expression levels of the c-Jun, Bcl-2 and Bcl-xL proteins, and levels of c-Jun protein phosphorylation were significantly decreased in SGC-BARF1 cells subsequent to treatment with SP600125, SB203580, and U0126, which were the specific inhibitors of JNK1/2/3, p38 and ERK1/2 respectively. In addition, there was a gradual increase in the protein expression and phosphorylation levels between normal gastric epithelial cells, and well-differentiated, moderately-differentiated and poorly-differentiated gastric carcinoma cells, but this was not statistically significant. Therefore, the present study hypothesized that JNK1/2/3-, p38- and ERK1/2-MAPK/c-Jun cascade signaling pathways may contribute to the upregulation of the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL induced by BARF1 in gastric carcinoma cells. This mechanism may mainly work in the progressive phase of the development in EBV-associated gastric carcinoma.

Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk).

Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog-sensitive (AS) kinases. We identified the gatekeeper residues of the v-Cdks encoded by Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) and mutated them to generate AS kinases. The AS-v-Cdks are functional and utilize different ATP derivatives with a specificity closely matching their cellular ortholog, AS-Cdk2. The AS derivative of the EBV v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral Cdks, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of both viral infections and Cdk biology.

Characterization of a Suppressive Cis-acting Element in the Epstein-Barr Virus LMP1 Promoter.

Latent membrane protein 1 (LMP1) is a major oncogene encoded by Epstein–Barr virus (EBV) and is essential for immortalization of B cells by the virus. Previous studies suggested that several transcription factors, such as PU.1, RBP-Jκ, NFκB, EBF1, AP-2 and STAT, are involved in LMP1 induction; however, the means by which the oncogene is negatively regulated remains unclear. Here, we introduced short mutations into the proximal LMP1 promoter that includes recognition sites for the E-box and Ikaros transcription factors in the context of EBV-bacterial artificial chromosome. Upon infection, the mutant exhibited increased LMP1 expression and EBV-mediated immortalization of B cells. However, single mutations of either the E-box or Ikaros sites had limited effects on LMP1 expression and transformation. Our results suggest that this region contains a suppressive cis-regulatory element, but other transcriptional repressors (apart from the E-box and Ikaros transcription factors) may remain to be discovered.

Latent Membrane Protein 1 Is a Novel Determinant of Epstein-Barr Virus Genome Persistence and Reactivation.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that persistently infects humans, with nearly 95% seropositivity in adults. Infection in differentiating epithelia is permissive, but EBV-associated nasopharyngeal carcinoma (NPC) tumors harbor a clonal and nonproductive latent infection. However, in explanted NPC cultures and epithelial cell lines, episomal EBV genomes are frequently lost. The resulting unstable infection has hampered efforts to study the determinants of EBV persistence and latency in epithelial oncogenesis. The EBV nuclear antigen 1 (EBNA1) protein is required for tethering EBV episomes to cellular DNA and for mitotic segregation to daughter cells. Expression of EBNA1 does not ensure faithful partitioning of EBV episomes or replicons, suggesting that additional regulatory mechanisms have yet to be elucidated. The EBV latent membrane protein 1 (LMP1) is an oncogenic signaling protein expressed in latent and lytic cycles. This study identified that LMP1 contributes to the loss of EBV genomes in latently infected cells and promotes differentiation-induced lytic replication in a polarized air-liquid interface (ALI) culture model. Deletion of LMP1 in recombinantly infected 293 cells promoted the retention of EBV genomes in passaged cells, which was in part localized to a conserved PXQXT motif in the C-terminal signaling domain (CTAR1). Additionally, knockdown of LMP1 in the recombinantly infected NPC cell line HK1 resulted in decreased induction of lytic proteins and infectious EBV titers. These findings are consistent with the hypothesis that in epithelial infections, regulation of LMP1 mechanisms may be a determinant of infection outcome and a potential risk factor for EBV persistence in preneoplastic cells. IMPORTANCE Latent membrane protein 1 (LMP1) is a constitutively active oncogenic signaling protein encoded by Epstein-Barr virus (EBV). Despite monoclonal infection in cases of nasopharyngeal carcinoma (NPC), it has been difficult to reconcile the heterogeneous LMP1 protein levels detected in tumor cells. The LMP1 protein is a pleiotropic signaling protein with oncogenic potential. Findings from this study are consistent with the hypothesis that LMP1 has a role distinct from that of oncogenesis that facilitates the viral life cycle by promoting an unstable but productive infection in differentiating epithelia.

Myalgic encephalomyelitis/chronic fatigue syndrome and gulf war illness patients exhibit increased humoral responses to the herpesviruses-encoded dUTPase: Implications in disease pathophysiology.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), and varicella-zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91-52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV-6 and human dUTPases than controls (P = 0.0053 and P = 0.0036, respectively), while the ME/CFS cohort had higher anti-EBV-dUTPase antibodies than in both GWI patients (P = 0.0008) and controls (P < 0.0001) as well as significantly higher anti-human dUTPase antibodies than in controls (P = 0.0241). These results suggest that screening of patients' sera for the presence of various combinations of anti-dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment.

Serum B-cell activating factor predicts prognosis in nasal type, extranodal NK/T cell lymphoma

B-cell activating factor (BAFF) plays important roles in a variety of lymphoid malignancies. Compared with healthy adults, patients with non-Hodgkin lymphoma had higher level of serum BAFF, and it corresponded with disease severity, response for therapy and clinical outcome. Latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) which is a known agent of nasal, extranodal NK/T cell lymphoma (ENKTCL) can switch the BAFF activating promoter leading to higher expression of BAFF in EBV-related tumor cells. However, the relationship between BAFF and ENKTCL has not been reported. Here we proposed a hypothesis that BAFF might play a regulatory role in ENKTCL development and maintenance. Our results showed that serum BAFF in ENKTCL patients was significantly higher than that in control group and negatively correlated with patients’ survival. It may be a valuable prognostic factor and deserved further study.

A locus encompassing the Epstein-Barr virus bglf4 kinase regulates expression of genes encoding viral structural proteins.

The mechanism regulating expression of late genes, encoding viral structural components, is an unresolved problem in the biology of DNA tumor viruses. Here we show that BGLF4, the only protein kinase encoded by Epstein-Barr virus (EBV), controls expression of late genes independent of its effect on viral DNA replication. Ectopic expression of BGLF4 in cells lacking the kinase gene stimulated the transcript levels of six late genes by 8- to 10-fold. Introduction of a BGLF4 mutant that eliminated its kinase activity did not stimulate late gene expression. In cells infected with wild-type EBV, siRNA to BGLF4 (siG4) markedly reduced late gene expression without compromising viral DNA replication. Synthesis of late products was restored upon expression of a form of BGLF4 resistant to the siRNA. Studying the EBV transcriptome using mRNA-seq during the late phase of the lytic cycle in the absence and presence of siG4 showed that BGLF4 controlled expression of 31 late genes. Analysis of the EBV transcriptome identified BGLF3 as a gene whose expression was reduced as a result of silencing BGLF4. Knockdown of BGLF3 markedly reduced late gene expression but had no effect on viral DNA replication or expression of BGLF4. Our findings reveal the presence of a late control locus encompassing BGLF3 and BGLF4 in the EBV genome, and provide evidence for the importance of both proteins in post-replication events that are necessary for expression of late genes.

Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells

The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes

All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC), which together facilitate the exit of assembled capsids from the nucleus. Previously, we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by the ORF67 and ORF69 genes when expressed in insect cells using baculoviruses for protein expression forms a complex at the nuclear membrane and remodels these membranes to generate nuclear membrane-derived vesicles. In this study, we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and remodeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67; however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type-membrane-anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation, but all abolish remodeling of nuclear membranes into circular structures. One mutant interacts with ORF67 as well as the wild-type protein but cannot function in membrane curvature and fission events that generate circular vesicles. These studies genetically confirm that ORF67 is required for cellular membrane proliferation and that ORF69 is the factor required to remodel these duplicated membranes into circular-virion-size vesicles. Furthermore, we also investigated the NEC encoded by Epstein-Barr virus (EBV). The EBV complex comprised of BFRF1 and BFLF2 was visualized at the nuclear membrane using autofluorescent protein fusions. BFRF1 is a potent inducer of membrane proliferation; however, BFLF2 cannot remodel these membranes into circular structures. What was evident is the superior remodeling activity of ORF69, which could convert the host membrane proliferations induced by BFRF1 into circular structures.

LMP1 and LMP2A are potential prognostic markers of extranodal NK/T-cell lymphoma, nasal type (ENKTL)

BackgroundLatent membrane protein (LMP) 1 and LMP2A encoded by Epstein-Barr virus (EBV) are associated with the development of malignancies, but their expression in extranodal NK/T-cell lymphoma, nasal type (ENKTL) and the relationship with clinical characteristics of this disease remain poorly understood. In the present study, we examined the expression of LMP1 and LMP2A in ENKTL, and investigated the correlations between LMP1 and LMP2A expression with clinicopathological characteristics of ENKTL patients.MethodsParaffin sections of surgically removed samples from 16 ENKTL patients were analyzed by immunohistochemistry and the related clinicopathological data were collected and analyzed.ResultsElevated expression (immunohistochemistry score ≥ 4) of LMP1 and LMP2A was detected in the tumor cells of ENKTL. High LMP1 expression was associated with positive B symptoms (p = 0.012), while high LMP2A expression was related to gender (p = 0.029). The expression of both LMP1 and LMP2A showed significant correlations with patients’ overall survival (p = 0.049, p = 0.036).ConclusionLMP1 and LMP2A may be prognostic indicators of survival in patients with ENKTL.Virtual slideshttp://www.diagnosticpathology.diagnomx.eu/vs/2443352538545899

Epstein-Barr virus oncoprotein LMP1 mediates survivin upregulation by p53 contributing to G1/S cell cycle progression in nasopharyngeal carcinoma

Latent membrane protein 1 (LMP1) is an important oncogenic protein encoded by Epstein-Barr virus (EBV) and plays an important role in the development of nasopharyngeal carcinoma (NPC). Our previous study has shown that p53 protein was accumulated and phosphorylated in NPC, implying its transcription factor activity in NPC tumorigenesis. However, the biological function and potential downstream target of p53 mediated by LMP1 in NPC remain unknown. In this study, we found that LMP1 simultaneously induced upregulation of both p53 and survivin at the protein level, as well as their phosphorylation. Knockdown of p53 by siRNA revealed that LMP1 increased survivin expression by p53 directly. Furthermore, we found that LMP1 upregulated survivin by p53 at the transcriptional level by increasing p53-mediated survivin promoter activity and DNA binding activity. Moreover, LMP1 induced the co-localization of p53 and survivin in the nucleus, conferring to their related functions in NPC tumorigenesis. We further found that p53 promoted G1/S cell cycle progression, but did not induce apoptosis in LMP1-positive NPC cells. Collectively, these findings suggest that p53 acting as a transcription factor promotes the transcriptional activity of survivin, and further increases its protein expression and phosphorylation in the regulation of LMP1, thus, leading to G1/S cell cycle progression with no effect on apoptosis in NPC tumorigenesis.

T7 phage displaying latent membrane protein 1 of Epstein-Barr virus elicits humoral and cellular immune responses in rats

The latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) has become a potential target in EBV-associated tumor prevention and treatment due to its multiple biological effects. In this study, the recombinant T7 phage displaying full-length LMP1 protein was cloned and used as an immunogen to immunize rats. Results of flow cytometry, Western blot analysis, and ELISA confirmed that both humoral and cellular immune responses were elicited in the immunized rats. Our data suggested that T7 phage was an efficient antigen carrier. The recombinant T7-LMP1 phage reconstitutes the antigenic and immunogenic properties of LMP1 and can serve as a vaccine against EBV.

Evaluation of LMP1 of Epstein-Barr virus as a therapeutic target by its inhibition

BackgroundThe latent membrane protein-1 (LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-κB. In so doing it leads to deregulated cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease. LMP1 is expressed in several EBV-associated malignancies, notably in Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). However, the viral protein is only detected in approximately 30%-50% of NPC samples, as such its role in carcinogenesis and tumour maintenance can be questioned and thus its relevance as a therapeutic target.ResultsIn order to explore if LMP1 has a continuous function in established tumours, its activity was inhibited through expression of a dominant negative LMP1 mutant in tumour cell lines derived from transgenic mice. LMP1 is the tumour predisposing oncogene in two different series of transgenic mice which separately give rise to either B-cell lymphomas or carcinomas. Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1. Inhibition of LMP1 activity in the transgenic B-cell lines was incompatible with growth and survival of the cells and no clones expressing the dominant negative LMP1 mutant could be established.ConclusionsLMP1 continues to provide a tumour cell growth function in cell lines established from LMP1 transgenic mouse tumours, of both B-cell and epithelial cell origin. LMP1 can perform this function, even when expressed at such low levels as to be undetectable, whereby evidence of its expression can only be inferred by its inhibition being detrimental to the growth of the cell. This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected. This reinforces the basis for pursuing LMP1 as a therapeutic target in EBV associated LMP1-expressing malignancies.

Epstein-Barr Virus Thymidine Kinase Is a Centrosomal Resident Precisely Localized to the Periphery of Centrioles

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

Modification of CɛmRNA 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 Cɛ 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. Cɛ mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cɛ mRNA levels were decreased in the splenocytes derived from the immunized rats. Cɛ mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.

Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line

Recently we confirmed that latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) accelerates a newly forming active c-Jun/Jun B heterodimer, a transcription factor, but little is known about the target gene regulated by it. In this paper, results indicated that a c-Jun/Jun B heterodimer induced by LMP1 upregulated cyclin D1 promoters activity and expression, on the contrary, downregulated p16, and maladjustment of cyclin D1 and p16 expression accelerated progression of cell cycle. Firstly, we found a c-Jun/Jun B heterodimer regulated synchronously and directly cyclin D1 and p16 in the Tet-on-LMP1-HNE2 cell line, in which LMP1 expression is regulated by Tet-on system. This paper investigated in depth function of the newly forming active c-Jun/Jun B heterodimer, and built new connection between environmental pathogenic factor, signal transduction and cell cycle.

Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis

The latent membrane protein (LMP1) encoded by Epstein-Barr virus (EBV) has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. RNA-cleaving DNA enzymes are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. In this study, we explore the potential of using DNAzymes as a therapeutic approach to EBV-associated carcinomas by targeting the LMP1 gene. In all, 13 different phosphorothioate-modified "10-23" deoxyribozymes (DNAzymes) were designed and synthesized against the LMP1 mRNA and transfected into B95-8 cells, which constitutively express the LMP1. Fluorescence microscopy was used to examine the cellular uptake and distribution in B95-8 cells. As demonstrated in Western blots, three out of 13 deoxyribozymes significantly downregulated the expression of LMP1 in B95-8 cells. These DNAzymes were shown to markedly inhibit B95-8 cell growth compared with a disabled DNAzyme and untreated controls, as determined by an alamarBlue Assay. It was further demonstrated that these DNAzymes arrested the B95-8 cells in G0/G1 using flow cytometry. Interestingly, the active DNAzymes could also downregulate the expression of Bcl-2 gene in treated cells, suggesting a close association between the LMP1 and Bcl-2 genes and their involvement in apoptosis. This was further confirmed with the result that the DNAzymes could induce the release of cytochrome c from mitochondria, which is the hallmark of the apoptosis. The present results suggest that the LMP1 may present a potential target for DNAzymes towards the EBV-associated carcinoma through cell proliferation and apoptosis pathways.

Latent membrane protein 1 of Epstein–Barr virus coordinately regulates proliferation with control of apoptosis

Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein-Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation.