RESUMO
Epstein-Barr virus (EBV) is a human oncogenic γ-herpesvirus that establishes persistent infection in more than 90% of the world's population. EBV has two life cycles, latency and lytic replication. Reactivation of EBV from latency to the lytic cycle is initiated and controlled by two viral immediate-early transcription factors, Zta and Rta, encoded by BZLF1 and BRLF1, respectively. In this study, we found that IQGAP2 expression was elevated in EBV-infected B cells and identified Rta as a viral gene responsible for the IQGAP2 upregulation in both B cells and nasopharyngeal carcinoma cell lines. Mechanistically, we showed that Rta increases IQGAP2 expression through direct binding to the Rta-responsive element in the IQGAP2 promoter. We also demonstrated the direct interaction between Rta and IQGAP2 as well as their colocalization in the nucleus. Functionally, we showed that the induced IQGAP2 is required for the Rta-mediated Rta promoter activation in the EBV lytic cycle progression and may influence lymphoblastoid cell line clumping morphology through regulating E-cadherin expression. IMPORTANCE Elevated levels of antibodies against EBV lytic proteins and increased EBV DNA copy numbers in the sera have been reported in patients suffering from Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, indicating that EBV lytic cycle progression may play an important role in the pathogenesis of EBV-associated diseases and highlighting the need for a more complete mechanistic understanding of the EBV lytic cycle. Rta acts as an essential transcriptional activator to induce lytic gene expression and thus trigger EBV reactivation. In this study, scaffolding protein IQGAP2 was found to be upregulated prominently following EBV infection via the direct binding of Rta to the RRE in the IQGAP2 promoter but not in response to other biological stimuli. Importantly, IQGAP2 was demonstrated to interact with Rta and promote the EBV lytic cycle progression. Suppression of IQGAP2 was also found to decrease E-cadherin expression and affect the clumping morphology of lymphoblastoid cell lines.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Proteínas Imediatamente Precoces , Neoplasias Nasofaríngeas , Humanos , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação ViralRESUMO
Whether and how a local virus infection affects the hematopoietic system in the bone marrow is largely unknown, unlike with systemic infection. In this study, we showed that influenza A virus (IAV) infection leads to demand-adapted monopoiesis in the bone marrow. The beta interferon (IFN-ß) promoter stimulator 1 (IPS-1)-type I IFN-IFN-α receptor 1 (IFNAR1) axis-mediated signaling was found to induce the emergency expansion of the granulocyte-monocyte progenitor (GMP) population and upregulate the expression of the macrophage colony-stimulating factor receptor (M-CSFR) on bipotent GMPs and monocyte progenitors via the signal transducer and activator of transcription 1 (STAT1), leading to a scaled-back proportion of granulocyte progenitors. To further address the influence of demand-adapted monopoiesis on IAV-induced secondary bacterial infection, IAV-infected wild-type (WT) and Stat1-/- mice were challenged with Streptococcus pneumoniae. Compared with WT mice, Stat1-/- mice did not demonstrate demand-adapted monopoiesis, had more infiltrating granulocytes, and were able to effectively eliminate the bacterial infection. IMPORTANCE Our findings show that influenza A virus infection induces type I interferon (IFN)-mediated emergency hematopoiesis to expand the GMP population in the bone marrow. The type I IFN-STAT1 axis was identified as being involved in mediating the viral-infection-driven demand-adapted monopoiesis by upregulating M-CSFR expression in the GMP population. As secondary bacterial infections often manifest during a viral infection and can lead to severe or even fatal clinical complications, we further assessed the impact of the observed monopoiesis on bacterial clearance. Our results suggest that the resulting decrease in the proportion of granulocytes may play a role in diminishing the IAV-infected host's ability to effectively clear secondary bacterial infection. Our findings not only provide a more complete picture of the modulatory functions of type I IFN but also highlight the need for a more comprehensive understanding of potential changes in hematopoiesis during local infections to better inform clinical interventions.
Assuntos
Interferon Tipo I , Infecções por Orthomyxoviridae , Receptor de Fator Estimulador de Colônias de Macrófagos , Fator de Transcrição STAT1 , Regulação para Cima , Animais , Humanos , Camundongos , Vírus da Influenza A/imunologia , Interferon Tipo I/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Infecções por Orthomyxoviridae/imunologia , Hematopoese/imunologia , Células Progenitoras de Granulócitos e Macrófagos/imunologia , Streptococcus pneumoniae/imunologia , Infecções Pneumocócicas/imunologiaRESUMO
The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet the underlying mechanisms explaining how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases (MAPKs) are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of MAPKs, during EBV infection and immortalization. Thirteen DUSPs, including 10 typical and 3 atypical types of DUSPs, were tested. Most of them were downregulated after EBV infection. Here, a role of viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates extracellular signal-regulated kinase (ERK) or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this report provides the first description of the interplay between EBV genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.IMPORTANCE Infections by the ubiquitous Epstein-Barr virus (EBV) are associated with a wide spectrum of lymphomas and carcinomas. It has been well documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual-specificity phosphates (DUSPs) exhibit great ability in regulating MAPK activities with respect to their capability of dephosphorylating MAPKs. In this study, we found that DUSPs were generally downregulated after EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppressed DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation was a continuous process. Furthermore, DUSP downregulation was found to contribute greatly to prevent apoptosis of EBV-infected cells. To sum up, this report sheds light on a novel molecular mechanism explaining how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.
Assuntos
Fosfatases de Especificidade Dupla/genética , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Apoptose/genética , Linfócitos B/virologia , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/metabolismo , Infecções por Vírus Epstein-Barr/virologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Virais/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Cultura Primária de Células , Proteínas da Matriz Viral/fisiologia , Proteínas Virais/metabolismo , Latência Viral/genética , Latência Viral/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin ß-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.
Assuntos
Núcleo Celular/virologia , Herpesvirus Humano 4/genética , Proteínas Virais/metabolismo , Liberação de Vírus/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Citoplasma/metabolismo , Glutationa Transferase/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Membrana Nuclear , Sinais de Localização Nuclear/metabolismo , Conformação Proteica , Análise de Sequência de Proteína , Proteínas Virais/química , Proteínas Virais/genética , Vírion/metabolismo , Liberação de Vírus/genética , beta CarioferinasRESUMO
During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.
Assuntos
Antígenos Virais/genética , DNA Helicases/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/metabolismo , Proteínas Imediatamente Precoces/genética , Glicoproteínas de Membrana/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Proteínas Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Replicação do DNA/genética , DNA Viral/genética , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.
Assuntos
Infecções por Vírus Epstein-Barr/complicações , Linfoma Difuso de Grandes Células B/mortalidade , Transtornos Linfoproliferativos/mortalidade , Receptor EphA4/metabolismo , Proteínas da Matriz Viral/metabolismo , Células Cultivadas , Regulação para Baixo , Infecções por Vírus Epstein-Barr/virologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Herpesvirus Humano 4 , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/virologia , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/virologia , Prognóstico , Receptor EphA4/genética , Transdução de Sinais , Taxa de Sobrevida , Proteínas da Matriz Viral/genéticaRESUMO
UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.
Assuntos
Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Células HeLa , HumanosRESUMO
Oncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II-related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease.
Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Transativadores/metabolismo , Fator 3 de Transcrição/genética , Proteínas da Matriz Viral/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Células Cultivadas , Regulação para Baixo , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Doença de Hodgkin/imunologia , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Técnicas Imunoenzimáticas , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/patologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 de Transcrição/metabolismo , Ativação Transcricional , Proteínas da Matriz Viral/genéticaRESUMO
BACKGROUND: Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. METHODS: We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. RESULTS: Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. CONCLUSION: Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.
Assuntos
Apigenina/farmacologia , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Proteínas Virais/metabolismo , Ativação Viral/efeitos dos fármacos , Linhagem Celular , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Humanos , Proteínas Virais/antagonistas & inibidoresRESUMO
UNLABELLED: BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin ß was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses. IMPORTANCE: During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.
Assuntos
Transporte Ativo do Núcleo Celular , Herpesvirus Humano 4/fisiologia , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Poro Nuclear/metabolismo , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
UNLABELLED: Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-κB, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-κB p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase Cδ (PKCδ) and inhibits its translocation from the cytoplasm to the nucleus, where PKCδ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification. IMPORTANCE: EBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-κB is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase Cδ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.
Assuntos
Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Interleucinas/biossíntese , Proteína Quinase C-delta/metabolismo , Proteínas da Matriz Viral/metabolismo , Latência Viral , Linfócitos B/virologia , Células Cultivadas , Herpesviridae , Humanos , Fator de Transcrição RelA/metabolismoRESUMO
UNLABELLED: Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE: Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , Herpesvirus Humano 4/genética , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Células HeLa , Herpesvirus Humano 4/metabolismo , Humanos , Replicação Viral/genéticaRESUMO
UNLABELLED: Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the third leftward open reading frame of the BamHI-A fragment in the viral genome, is a homologue of herpes simplex virus type 1 UL28. This gene product is believed to possess the properties of a terminase, such as nucleolytic activity on newly synthesized viral DNA and translocation of unit length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced by and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, which determined that EBV BALF3 acts as an endonuclease and its activity is modulated by Mg(2+), Mn(2+), and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following induction of the lytic cycle, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the basis of these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions. IMPORTANCE: Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of the human herpesvirus Epstein-Barr virus (EBV), BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence on the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in contributing to a better understanding of the EBV life cycle.
Assuntos
Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Cátions Bivalentes/metabolismo , Ativadores de Enzimas/metabolismo , Magnésio/metabolismo , Manganês/metabolismoRESUMO
(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and ß-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and ß-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.
Assuntos
Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Gelatinases/metabolismo , Animais , Caderinas/metabolismo , Carcinoma , Caspase 3/metabolismo , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Transplante Heterólogo , Regulação para Cima/efeitos dos fármacosRESUMO
Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.
Assuntos
Linfócitos B/virologia , Proliferação de Células , Quimiocina CCL3/biossíntese , Quimiocina CCL4/biossíntese , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas da Matriz Viral/metabolismo , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
Assuntos
Núcleo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Herpesvirus Humano 4/fisiologia , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Ligação Proteica/genética , Transporte Proteico , Distribuição Tecidual , Transfecção , Proteínas Virais/genética , Proteínas Virais/fisiologia , Montagem de Vírus/genética , Liberação de Vírus/genética , Liberação de Vírus/fisiologiaRESUMO
BACKGROUND: Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection. RESULTS: We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/ß receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2-/- mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice. CONCLUSIONS: Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.
Assuntos
Quimiocinas/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/fisiopatologia , Infecções por Orthomyxoviridae/fisiopatologia , Receptor de Interferon alfa e beta/genética , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Infecções por Orthomyxoviridae/virologia , Oseltamivir/farmacologia , Receptor de Interferon alfa e beta/metabolismo , Receptores CCR2/metabolismo , Índice de Gravidade de Doença , Organismos Livres de Patógenos Específicos , Replicação Viral/efeitos dos fármacosRESUMO
Epstein-Barr virus (EBV) BGLF4 is a member of the conserved herpesvirus kinases that regulate multiple cellular and viral substrates and play an important role in the viral lytic cycles. BGLF4 has been found to phosphorylate several cellular and viral transcription factors, modulate their activities, and regulate downstream events. In this study, we identify an NF-κB coactivator, UXT, as a substrate of BGLF4. BGLF4 downregulates not only NF-κB transactivation in reporter assays in response to tumor necrosis factor alpha (TNF-α) and poly(I·C) stimulation, but also NF-κB-regulated cellular gene expression. Furthermore, BGLF4 attenuates NF-κB-mediated repression of the EBV lytic transactivators, Zta and Rta. In EBV-positive NA cells, knockdown of BGLF4 during lytic progression elevates NF-κB activity and downregulates the activity of the EBV oriLyt BHLF1 promoter, which is the first promoter activated upon lytic switch. We show that BGLF4 phosphorylates UXT at the Thr3 residue. This modification interferes with the interaction between UXT and NF-κB. The data also indicate that BGLF4 reduces the interaction between UXT and NF-κB and attenuates NF-κB enhanceosome activity. Upon infection with short hairpin RNA (shRNA) lentivirus to knock down UXT, a spontaneous lytic cycle was observed in NA cells, suggesting UXT is required for maintenance of EBV latency. Overexpression of wild-type, but not phosphorylation-deficient, UXT enhances the expression of lytic proteins both in control and UXT knockdown cells. Taking the data together, transcription involving UXT may also be important for EBV lytic protein expression, whereas BGLF4-mediated phosphorylation of UXT at Thr3 plays a critical role in promoting the lytic cycle.
Assuntos
Regulação para Baixo , Regulação Viral da Expressão Gênica , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Proteínas de Ciclo Celular , Células HEK293 , Células HeLa , Humanos , Lentivirus/genética , Chaperonas Moleculares , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Nasopharyngeal carcinoma (NPC) is characteristic for its strong association with Epstein-Barr virus (EBV) and high metastatic rate. Recently, overexpressed recepteur d'origine nantais (RON) (MST1R), receptor tyrosine kinase has been reported in human cancers and tumor metastasis. Therefore, the role of RON in EBV-associated NPC and its metastasis was investigated. Here we show that RON was found in NPC but not in control tissues. A significant correlation of latent membrane protein 1 (LMP1) and RON expression was found in NPC (Pearson's χ(2) test; P = 0.0023). At the molecular level, LMP1 stimulates nuclear factor-κB binding to the RON promoter through its carboxyl-terminal activation region 1 to induce expression of RON. Knockdown of RON in cells expressing LMP1 significantly reverses LMP1-induced epithelial-mesenchymal transition and suppresses LMP1-induced cell migration and invasion. These results suggest an important role of RON in the tumorigenesis and metastasis of NPC and RON may be a novel therapeutic target for EBV-associated NPC.
Assuntos
Infecções por Vírus Epstein-Barr/enzimologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/virologia , Receptores Proteína Tirosina Quinases/metabolismo , Carcinoma , Movimento Celular , Forma Celular , Transição Epitelial-Mesenquimal , Infecções por Vírus Epstein-Barr/patologia , Humanos , Imuno-Histoquímica , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Transdução de Sinais , Proteínas da Matriz Viral/metabolismoRESUMO
EBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.