村田 貴之

Epigenetic modifications play a pivotal role in the expression of the genes of Epstein-Barr virus (EBV). We found that de novo EBV infection of primary B cells caused moderate induction of enhancer of zeste homolog 2 (EZH2), the major histone H3 lysine 27 (K27) methyltransferase. To investigate the role of EZH2, we knocked out the EZH2 gene in EBV-negative Akata cells by the CRISPR/Cas9 system and infected the cells with EBV, followed by selection of EBV-positive cells. During the latent state, growth of EZH2-knockout (KO) cells was significantly slower after infection compared to wild-type controls, despite similar levels of viral gene expression between cell lines. After induction of the lytic cycle by anti-IgG, KO of EZH2 caused notable induction of expression of both latent and lytic viral genes, as well as increases in both viral DNA replication and progeny production. These results demonstrate that EZH2 is crucial for the intricate epigenetic regulation of not only lytic but also latent gene expression in Akata cells.IMPORTANCE The life cycle of EBV is regulated by epigenetic modifications, such as CpG methylation and histone modifications. Here, we found that the expression of EZH2, which encodes a histone H3K27 methyltransferase, was induced by EBV infection; therefore, we generated EZH2-KO cells to investigate the role of EZH2 in EBV-infected Akata B cells. Disruption of EZH2 resulted in increased expression of EBV genes during the lytic phase and, therefore, efficient viral replication and progeny production. Our results shed light on the mechanisms underlying reactivation from an epigenetic point of view and further suggest a role for EZH2 as a form of innate immunity that restricts viral replication in infected cells.

An unusual rotavirus strain, DB2015-066 with the G10P[14] genotype (RVA/Human-wt/THA/DB2015-066/2015/G10P[14]), was detected in a stool sample from a child hospitalized with acute gastroenteritis in Thailand. Here, we sequenced and characterized the full-genome of the strain DB2015-066. On whole genomic analysis, strain DB2015-066 was shown to have a unique genotype constellation: G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The backbone genes of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) are commonly found in rotavirus strains from artiodactyls such as cattle. Furthermore, phylogenetic analysis indicated that each of the 11 genes of strain DB2015-066 could be of artiodactyl (likely bovine) origin. Thus, strain DB2015-066 appeared to be derived from through zoonotic transmission of a bovine rotavirus strain. Of note, the VP7 gene of strain DB2015-066 was located in G10 lineage-6 together with ones of bovine and bovine-like rotavirus strains, away from the clusters comprising other G10P[14] strains in G10 lineage-2/4/5/9, suggesting the occurrence of independent bovine-to-human interspecies transmission events. Our observations provide important insights into the origins of rare G10P[14] strains, and into dynamic interactions between artiodactyl and human rotavirus strains.

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors.

In addition to latent genes, lytic genes of EBV must also be of extreme significance since propagation of the virus can be achieved only through execution of lytic cycle. Research on EBV lytic genes may thus prevent spreading of the virus and alleviate disorders, such as infectious mononucleosis and oral hairy leukoplakia, which are highly associated with EBV lytic infection. Moreover, recent advancements have been demonstrating that at least several lytic genes are expressed to some extent even during latent state. It is also demonstrated now that upon de novo infection, EBV expresses lytic genes in addition to latent genes before establishment of latency (this phase is called “pre-latent abortive lytic state”). In those cases, lytic genes also play important roles in cell proliferation of EBV-positive cells. However, many lytic gene products have not been identified yet nor studied thoroughly, and even worse, some have been misidentified in the literature. Here, I would like to give a detailed up-to-date review on EBV lytic genes.

Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

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-J kappa, NF kappa B, 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.

The Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with several malignancies. It establishes a latent infection in B lymphocytes and is occasionally reactivated to enter the lytic cycle. Here we examined the role of the EBV gene BRRF1, which is expressed in the lytic state. We first confirmed, using a DNA polymerase inhibitor, that the BRRF1 gene is expressed with early kinetics. A BRRF1-deficient recombinant virus was constructed using a bacterial artificial chromosome system. No obvious differences were observed between the wild-type, BRRF1-deficient mutant and the revertant virus in HEK293 cells in terms of viral lytic protein expression, viral DNA synthesis, progeny production, pre-latent abortive lytic gene expression and transformation of primary B cells. However, reporter assays indicated that BRRF1 may activate transcription in promoter-and cell type-dependent manners. Taken together, BRRF1 is dispensable for viral replication in HEK293 cells and transformation of B cells, but it may have effects on transcription.

Epstein-Barr virus (EBV) latently infects malignant epithelial cells in approximately 10% of all gastric cancers. Latent membrane protein 1 (LMP1), an oncogenic protein, plays an important role in malignant transformation in EBV-associated nasopharyngeal carcinoma and B-cell lymphoma; however, its expression has not been detected in EBV-associated gastric cancer. To address why LMP1 has not been detected in EBV-positive gastric tumors, we focused on the interactions between LMP1-positive and -negative cells and stably expressed LMP1 in the gastric cancer cell line AGS. We showed that the number of LMP1-positive cells decreased gradually with each cell passage when the cells were co-cultured with LMP1-negative cells. Time-lapse imaging showed that LMP1-positive cells were eliminated from a monolayer of LMP1-negative cells. Furthermore, LMP1-positive cells stimulated the proliferation of surrounding LMP1-negative cells, but not LMP1-positive cells, via exosome-mediated EGFR activation. Our data indicate that LMP1 expression drives cell competition between LMP1-positive and -negative cells, affecting the behavior of the cells within gastric tissue.

Inflammasomes are cytoplasmic sensors that regulate the activity of caspase-1 and the secretion of interleukin-1 beta (IL-1 beta) or interleukin-18 (IL-18) in response to foreign molecules, including viral pathogens. They are considered to be an important link between the innate and adaptive immune responses. However, the mechanism by which inflammasome activation occurs during primary Epstein-Barr virus (EBV) infection remains unknown. Human B lymphocytes and epithelial cells are major targets of EBV, although it can also infect a variety of other cell types. In this study, we found that EBV could infect primary human monocytes and the monocyte cell line, THP-1, inducing inflammasome activation. We incubated cell-free EBV with THP-1 cells or primary human monocytes, then confirmed EBV infection using confocal microscopy and flow cytometry. Lytic and latent EBV genes were detected by real-time RT-PCR in EBV-infected monocytes. EBV infection of THP-1 cells and primary human monocytes induced caspase-dependent IL-1 beta production, while EBV infection of B-cell or T-cell lines did not induce IL-1 beta production. To identify the sensor molecule responsible for inflammasome activation during EBV infection, we examined the mRNA and the protein levels of NLR family pyrin domain-containing 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon-inducible protein 16 (IFI16). Increased AIM2 levels were observed in EBV-infected THP-1 cells and primary human monocytes, whereas levels of IFI16 and NLRP3 did not show remarkable change. Furthermore, knockdown of AIM2 by small interfering RNA attenuated caspase-1 activation. Taken together, our results suggest that EBV infection of human monocytes induces caspase-1-dependent IL-1 beta production, and that AIM2, acting as an inflammasome, is involved in this response.

Advanced melanoma has long been treated with chemotherapy using cytotoxic agents like dacarbazine (DTIC), but overall survival rates with these drugs have been generally low. Recently, immunoregulatory monoclonal antibodies and molecularly targeted therapy with a BRAF inhibitor and/or a MEK inhibitor, have been used to treat malignant melanoma and have improved the survival rate of patients with advanced melanoma. However, high prices of these drugs are problematic. In this study, we evaluated the oncolytic efficacy of HF10, an attenuated, replication-competent HSV, with DTIC in immunocompetent mice model of malignant melanoma. For in vitro studies, cytotoxicity assays were conducted in clone M3 mouse melanoma cells. For the in vivo studies, subcutaneous melanoma models were prepared in DBA/2 mice with clone M3 cells, and then HF10 was intratumorally inoculated with/without intraperitoneal DTIC injection. The efficacy of the therapies was evaluated by survival, growth of subcutaneous tumor, and histopathological and immunological analyses. Both HF10 infection and DTIC treatment showed cytotoxic effects in melanoma cells, but combination treatment with HF10 and DTIC showed a rapid and strong cytotoxic effect compared with monotherapy. In the subcutaneous melanoma model, intratumoral HF10 inoculation significantly inhibited tumor growth. HF10 also inhibited the growth of non-inoculated contralateral tumors when it was injected into the ipsilateral tumors of mice. In histologic and immunohistochemical analysis, tumor lysis and inflammatory cell infiltration were observed after intratumoral HF10 inoculation. When mice were treated with HF10 and DTIC, the combination therapy induced a robust systemic anti-tumor immune response and prolonged survival. IFN-gamma secretion from splenocytes of the HF10-DTIC combination therapy group showed more IFN-gamma secretion than did the other groups. These data showed the efficacy of HF10 and DTIC combination therapy in a mouse melanoma model.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with several malignancies. We reported previously that an EBV lytic gene product BRRF2 is involved in the maturation of progeny virus. To analyze the domain(s) needed for efficient production of progeny, we prepared a series of deletion mutants and found two functional domains in the N- and C-terminal regions by complementation assays. Immunofluorescence analyses revealed that BRRF2 lacking the C-terminal region demonstrated aberrant localization in both the nucleus and cytoplasm, whereas wild-type BRRF2 was localized predominantly in the cytoplasm. We also confirmed that wild-type BRRF2 co-localized with Rab5, an endosomal marker, at least partly. Additionally, serine 511 of BRRF2 was phosphorylated during lytic infection; however, a mutant in which the serine was substituted with alanine still augmented the yield as efficiently as did wild-type BRRF2. These results showed that the C-terminal region of BRRF2 is involved in the predominant localization of BRRF2 to the cytoplasm and in the efficient production of infectious virus.

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-J kappa, NF-kappa B, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1.

The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, and occasionally switches from the latent state to the lytic cycle. In this report, we identified and examined the role of a lytic gene, BRRF2. We first prepared an antibody against BRRF2 and identified the gene product as a viral lytic protein expressed in B95-8 cells with late kinetics. Immunofluorescence revealed that BRRF2 localized in the cytoplasm of cells during the lytic phase. We also found that BRRF2 protein was phosphorylated in lytic cells, but the only viral protein kinase, BGLF4, was not involved in the phosphorylation. Knockout EBV and a repaired strain were then prepared, and we found that BRRF2 disruption did not affect viral gene expression and DNA replication, but decreased virus production. These results demonstrated that BRRF2 is involved in production of infectious progeny, although it is not essential for lytic replication. (C) 2015 Elsevier Inc. All rights reserved.

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, but a small percentage of infected cells switch from the latent state to the lytic cycle, leading to potent viral DNA replication and progeny viruses production. We here focused on a lytic gene BGLF3.5, and first established BGLF3.5 mutants by marker cassette insertion. Unexpectedly, this insertion mutant failed to produce BGLF4 protein and thus progeny production was severely inhibited. Then we carefully made two point mutant viruses (stop codon insertion or frame-shift mutation) and found that BGLF3.5 is not essential for EBV lytic replication processes, such as viral gene expression, DNA replication, or progeny production in the HEK293 cells although its homolog in murine gammaherpesvirus 68 (MHV-68) was reported to be essential. In addition, we examined the roles of two short, upstream open reading frames within the 5'UTR of BGLF3.5 gene in translation of BGLF4. (C) 2015 Elsevier Inc. All rights reserved.

Epstein-Barr virus (EBV) is associated with several malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. To overcome such disorders, understanding the molecular mechanisms of the EBV replication is important. The EBV DNA polymerase (Pol) is one of the essential factors for viral lytic DNA replication. Although it is well known that its C-terminal half, possessing DNA polymerase and 3'-5' exonuclease activity, is highly conserved among Family B Pols, the NH2-terminal half has yet to be characterized in detail. In this study, we show that a stretch of hydrophobic amino acids within the pre-NH2-terminal domain of EBV Pol plays important role. In addition, we could identify the most essential residue for replication in the motif. These findings will shed light on molecular mechanisms of viral DNA synthesis and will help to develop new herpesviruses treatments.

Epstein-Barr virus (EBV), which infects not only B cells but also T and natural killer (NK) cells, is associated with a variety of lymphoid malignancies. Because EBV-associated T and NK cell lymphomas are refractory and resistant to conventional chemotherapy, there is a continuing need for new effective therapies. EBV-encoded "latent membrane protein 1" (LMP1) is a major oncogene that activates nuclear factor kappa B (NF-kappa B), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase signaling pathways, thus promoting cell growth and inhibiting apoptosis. Recently, we screened a library of small-molecule inhibitors and isolated heat shock protein 90 (Hsp90) inhibitors as candidate suppressors of LMP1 expression. In this study, we evaluated the effects of BIIB021, a synthetic Hsp90 inhibitor, against EBV-positive and -negative T and NK lymphoma cell lines. BIIB021 decreased the expression of LMP1 and its downstream signaling proteins, NF-kappa B, JNK, and Akt, in EBV-positive cell lines. Treatment with BIIB021 suppressed proliferation in multiple cell lines, although there was no difference between the EBV-positive and -negative lines. BIIB021 also induced apoptosis and arrested the cell cycle at G1 or G2. Further, it down-regulated the protein levels of CDK1, CDK2, and cyclin D3. Finally, we evaluated the in vivo effects of the drug; BIIB021 inhibited the growth of EBV-positive NK cell lymphomas in a murine xenograft model. These results suggest that BIIB021 has suppressive effects against T and NK lymphoma cells through the induction of apoptosis or a cell cycle arrest. Moreover, BIIB021 might help to suppress EBV-positive T or NK cell lymphomas via the down-regulation of LMP1 expression.

Purpose: Epstein-Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells, and T- and NK-cell lymphoproliferative diseases (T/NK-LPD) that are refractory to conventional chemotherapies may develop. To identify a molecular-targeted therapy for EBV-associated T/NK-LPDs, we investigated whether CC chemokine receptor 4 (CCR4) was expressed on EBV-infected T and/or NK cells and whether a humanized anti-CCR4 monoclonal antibody, mogamulizumab, was effective. Experimental Design: CCR4 expression was examined in various cell lines. In vitro, the effects of mogamulizumab on cell lines were evaluated in the presence of peripheral blood mononuclear cells from volunteers. In vivo, the effects of mogamulizumab were evaluated using a murine xenograft model. CCR4 expression was examined on EBV-infected cells from patients with EBV-associated T/NK-LPDs. Ex vivo, the effects of mogamulizumab were evaluated using patient lymphocytes. Results: CCR4 expression was confirmed in most EBV-positive T and NK cell lines. Mogamulizumab induced antibody-dependent cellular cytotoxicity (ADCC) activity against CCR4-positive cell lines, and inhibited the growth of EBV-positive NK-cell lymphomas in a murine xenograft model. Furthermore, CCR4 was expressed on EBV-infected cells in 8 of 17 patients with EBV-associated T/NK-LPDs. Interestingly, CCR4 was positive in 5 of 5 patients with hydroa vacciniforme, a photodermatosis caused by the clonal expansion of EBV-infected gamma delta T cells. EBV-positive gamma delta T cells were obtained from a patient with hydroa vacciniforme and subjected to an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. The gamma delta T cells that were positive for CCR4 were killed by mogamulizumab via ADCC. Conclusions: These results indicate that mogamulizumab may be a therapeutic option against EBV-associated T/NK-LPDs. (C)2014 AACR.

Epstein-Barr virus (EBV) predominantly infects B cells and causes B-cell lymphomas, such as Burkitt lymphoma and Hodgkin lymphoma. However, it also infects other types of cells, including T and natural killer (NK) cells, and causes disorders, such as chronic active EBV infection (CAEBV) and T/NK-cell lymphoma. The CAEBV is a lymphoproliferative disease with poor prognosis, where EBV-positive T or NK cells grow rapidly, although the molecular mechanisms that cause the cell expansion still remain to be elucidated. EBV-encoded latent membrane protein 1 (LMP1) is an oncogene that can transform some cell types, such as B cells and mouse fibroblasts, and thus may stimulate cell proliferation in CAEBV. Here, we examined the effect of LMP1 on EBV-negative cells using the cells conditionally expressing LMP1, and on CAEBV-derived EBV-positive cells by inhibiting the function of LMP1 using a dominant negative form of LMP1. We demonstrated that LMP1 was responsible for the increased cell proliferation in the cell lines derived from CAEBV, while LMP1 did not give any proliferative advantage to the EBV-negative cell line.

The EBV is a human gamma-herpesvirus associated with various neoplasms. It is responsible for causing cancers of B, T, and NK cells as well as cells of epithelial origin. Such diversity in target cells and the complicated steps of oncogenesis are perplexing when we speculate about the mechanisms of action of EBV-positive cancers. Here, we first note three common features that contribute to the development and maintenance of EBV-positive cancers: effects of EBV oncogenes, immunosuppression and evasion/exploitation of the immune system, and genetic and epigenetic predisposition/alteration of the host genome. Then, we demonstrate the mechanisms of oncogenesis and the means by which each EBV-positive cancer develops, with particular focus on the mode of EBV infection. The EBV has two alternative life cycles: lytic and latent. The latter is categorized into four programs ( latency types 0-III) in which latent viral genes are expressed differentially depending on the tissue of origin and state of cells. The production of viral latent genes tends to decrease with an increase in time, and, in an approximate manner, the expression levels of viral genes are inversely correlated with the degree of abnormalities in the host genome. Occasional execution of the viral lytic cycle also contributes to oncogenesis. Understanding this life cycle of the EBV and its relevance in oncogenesis may provide valuable clues to the development of effective therapies for the associated cancers. Copyright (C) 2014 John Wiley & Sons, Ltd.

The ubiquitous Epstein-Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells and is associated with various lymphoid malignancies. Recent studies have reported that histone deacetylase (HDAC) inhibitors exert anticancer effects against various tumor cells. In the present study, we have evaluated both the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on EBV-positive and EBV-negative T and NK lymphoma cells. Several EBV-positive and EBV-negative T and NK cell lines were treated with various concentrations of SAHA. SAHA suppressed the proliferation of T and NK cell lines, although no significant difference was observed between EBV-positive and EBV-negative cell lines. SAHA induced apoptosis and/or cell cycle arrest in several T and NK cell lines. In addition, SAHA increased the expression of EBV-lytic genes and decreased the expression of EBV-latent genes. Next, EBV-positive NK cell lymphoma cells were subcutaneously inoculated into severely immunodeficient NOD/Shi-scid/IL-2Rnull mice, and then SAHA was administered intraperitoneally. SAHA inhibited tumor progression and metastasis in the murine xenograft model. SAHA displayed a marked suppressive effect against EBV-associated T and NK cell lymphomas through either induction of apoptosis or cell cycle arrest, and may represent an alternative treatment option.

The Epstein-Barr virus (EBV) is a human gamma-herpesvirus that is implicated in various types of proliferative diseases. Upon infection, it predominantly establishes latency in B cells and cannot ever be eradicated; it persists for the host's lifetime. Reactivation of the virus from latency depends on expression of the viral immediate-early gene, BamHI Z fragment leftward open reading frame 1 (BZLF1). The BZLF1 promoter normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore, histone deacetylase inhibitor, or anti-Ig. Transcription from the BZLF1 promoter is activated by myocyte enhancer factor 2, specificity protein 1, b-Zip type transcription factors and mediating epigenetic modifications of the promoter, such as histone acetylation and H3K4me3. In contrast, repression of the promoter is mediated by transcriptional suppressors, such as ZEB, ZIIR-BP, and jun dimerization protein 2, causing suppressive histone modifications like histone H3K27me3, H3K9me2/3 and H4K20me3. Interestingly, there is little CpG DNA methylation of the promoter, indicating that DNA methylation is not crucial for suppression of BZLF1. This review will focus on the molecular mechanisms by which the EBV lytic switch is controlled and discuss the physiological significance of this switching for its survival and oncogenesis.

The EBV is a human gamma-herpesvirus that is associated with a variety of neoplasms. Upon primary infection, it transiently runs a short lytic program and then predominantly establishes latent infection. Only a small percentage of infected cells switch from the latent stage into the lytic cycle and produce progeny viruses. Although EBV in cancer cells is mostly in the latent state, the lytic cycle of the virus is also expected to play a pivotal role in development and maintenance of tumors because of its association with secretion of cytokines or growth factors. Moreover, if efficient artificial induction of lytic replication could somehow be achieved, development of oncolytic therapy for EBV-positive cancers would be conceivable. Thus, understanding the switching mechanism is of essential importance. Reactivation of the virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore, or histone deacetylase inhibitors. Transcription from the Zp is regulated by the balance between active and suppressive epigenetic histone marks, including histone acetylation, histone H3 Lysine 4 trimethylation and histone H3 lysine 27 trimethylation, being mediated by multiple transcription factors, such as myocyte enhancer factor 2, specificity protein 1, and zinc finger E-box binding homeobox. This review will focus on such molecular mechanisms by which the EBV lytic switch is controlled and discuss the physiological significance of the switching for oncogenesis. Copyright (c) 2013 John Wiley & Sons, Ltd.

Epstein-Barr virus (EBV) is a member of gamma-herpesvirus, which can cause various types of tumor. Coexisting with the host for a long period of time, it has evolved unique and sophisticated strategy for survival by taking complicated, tactical modes of infection. Such modes include latent and lytic infections, and latent state is further categorized into four types. Differences and transitions in such lifestyles are significantly associated not only with virus amplification, but also with pathology and advancement of the disorders. I here review oncogenesis and pathogenesis of EBV-related disorders, especially focusing on our recent results on the modes of EBV infection.

Reactivation of Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including those encoding MEF2B, KLF4, and some cellular b-Zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we prepared recombinant EBV-BAC, in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293 cells. The virus failed to induce a subset of early genes, such as that encoding BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could be completely lifted by exogenous supply of BRLF1, together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1, but also BRLF1 in comparison to wild-type or revertant virus cases. Wealso observed that MEF2 mutant EBV featured relatively high repressive histone methylation, such as H3K27me3, but CpGDNAmethylation levels were comparable around Zp and the BRLF1 promoter (Rp). These findings shed light on BZLF1 expression and EBV reactivation from latency.

Background: Epstein-Barr virus episomes piggyback onto cellular chromosomes in latently infected cells. Results: EBNA1 was functionally sound even when arginines within its chromosome binding domains were replaced with lysines. Conclusion: The basic nature of the domains is critically important for the chromosome binding. Significance: Learning how EBNA1 attaches to chromosomes is crucial for understanding the mechanism of stable maintenance of viral episomes. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Epstein-Barr virus (EBV) replication proteins are transported into the nucleus to synthesize viral genomes. We here report molecular mechanisms for nuclear transport of EBV DNA polymerase. The EBV DNA polymerase catalytic subunit BALF5 was found to accumulate in the cytoplasm when expressed alone, while the EBV DNA polymerase processivity factor BMRF1 moved into the nucleus by itself. Coexpression of both proteins, however, resulted in efficient nuclear transport of BALF5. Deletion of the nuclear localization signal of BMRF1 diminished the proteins' nuclear transport, although both proteins can still interact. These results suggest that BALF5 interacts with BMRF1 to effect transport into the nucleus. Interestingly, we found that Hsp90 inhibitors or knockdown of Hsp90β with short hairpin RNA prevented the BALF5 nuclear transport, even in the presence of BMRF1, both in transfection assays and in the context of lytic replication. Immunoprecipitation analyses suggested that the molecular chaperone Hsp90 interacts with BALF5. Treatment with Hsp90 inhibitors blocked viral DNA replication almost completely during lytic infection, and knockdown of Hsp90β reduced viral genome synthesis. Collectively, we speculate that Hsp90 interacts with BALF5 in the cytoplasm to assist complex formation with BMRF1, leading to nuclear transport. Hsp90 inhibitors may be useful for therapy for EBV-associated diseases in the future. © 2013, American Society for Microbiology.

Productive replication of the Epstein-Barr virus (EBV) occurs in discrete sites in nuclei, called replication compartments, where viral genome DNA synthesis and transcription take place. The replication compartments include subnuclear domains, designated BMRF1 cores, which are highly enriched in the BMRF1 protein. During viral lytic replication, newly synthesized viral DNA genomes are organized around and then stored inside BMRF1 cores. Here, we examined spatial distribution of viral early and late gene mRNAs within replication compartments using confocal laser scanning microscopy and three-dimensional surface reconstruction imaging. EBV early mRNAs were mainly located outside the BMRF1 cores, while viral late mRNAs were identified inside, corresponding well with the fact that late gene transcription is dependent on viral DNA replication. From these results, we speculate that sites for viral early and late gene transcription are separated with reference to BMRF1 cores.

Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFkB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immunodefficient NOD/Shi-scid/IL-2R gamma(null) (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication©2013, American Society for Microbiology.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.

The Epstein-Barr virus (EBV) predominantly establishes latent infection in B cells, and the reactivation of the virus from latency is dependent on the expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores, or histone deacetylase (HDAC) inhibitors. In some cell lines latently infected with EBV, an HDAC inhibitor alone can induce BZLF1 transcription, while the treatment does not enhance expression in other cell lines, such as B95-8 or Raji cells, suggesting unknown suppressive mechanisms besides histone deacetylation in those cells. Here, we found the epigenetic modification of the BZLF1 promoter in latent Raji cells by histone H3 lysine 27 trimethylation (H3K27me3), H3K9me2/me3, and H4K20me3. Levels of active markers such as histone acetylation and H3K4me3 were low in latent cells but increased upon reactivation. Treatment with 3-deazaneplanocin A (DZNep), an inhibitor of H3K27me3 and H4K20me3, significantly enhanced the BZLF1 transcription in Raji cells when in combination with an HDAC inhibitor, trichostatin A (TSA). The knockdown of Ezh2 or Suv420h1, histone methyltransferases for H3K27me3 or H4K20me3, respectively, further proved the suppression of Zp by the methylations. Taken together, the results indicate that H3K27 methylation and H4K20 methylation are involved, at least partly, in the maintenance of latency, and histone acetylation and H3K4 methylation correlate with the reactivation of the virus in Raji cells.

Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-kappa B, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBP epsilon as a new transcriptional activator. Exogenous expression of C/EBP alpha, -beta, or -epsilon efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.

A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akata-derived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics.

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

Productive replication of Epstein-Barr virus occurs in discrete sites in nuclei, called replication compartments, where viral DNA replication proteins and host homologous recombinational repair (HRR) and mismatch repair (MMR) factors are recruited. Three-dimensional (3D) surface reconstruction imaging clarified the spatial arrangements of these factors within the replication compartments. Subnuclear domains, designated BMRF1 cores, which were highly enriched in viral polymerase processivity factor BMRF1 could be identified inside the replication compartments. Pulse-chase experiments revealed that newly synthesized viral genomes organized around the BMRF1 cores were transferred inward. HRR factors could be demonstrated mainly outside BMRF1 cores, where de novo synthesis of viral DNA was ongoing, whereas MMR factors were found predominantly inside. These results imply that de novo synthesis of viral DNA is coupled with HRR outside the cores, followed by MMR inside cores for quality control of replicated viral genomes. Thus, our approach unveiled a viral genome manufacturing plant.

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the BZLF1 viral immediate-early protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found that Jun dimerization protein 2 (JDP2) plays a significant role in suppressing Zp activity. Reporter, EMSA, and ChIP assays of a Zp mutant virus revealed JDP2 association with Zp at the ZII cis-element, a binding site for CREB/ATF/AP-1. Suppression of Zp activity by JDP2 correlated with HDAC3 association and reduced levels of histone acetylation. Although introduction of point mutations into the ZII element of the viral genome did not increase the level of BZLF1 production, silencing of endogenous JDP2 gene expression by RNA interference increased the levels of viral early gene products and viral DNA replication. These results indicate that JDP2 plays a role as a repressor of Zp and that its replacement by CREB/ATF/AP-1 at ZII is crucial to triggering reactivation from latency to lytic replication.