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N6-methyladenosine (m6A) mediates various biological processes by affecting RNA stability, splicing, and translational efficiency. The roles of m6A modification in Epstein-Barr virus (EBV) infection in the lytic phase are unclear. Here, knockout of the m6A methyltransferase, N6-methyladenosine methyltransferase-like 3 (METTL3), or inhibition of methylation by DAA or UZH1a decreased the expression of viral lytic proteins and reduced progeny virion production. Interestingly, cell growth and viability were decreased by induction of the lytic cycle in METTL3-knockout or inhibitor-treated cells. Apoptosis was induced in those conditions possibly because of a decreased level of the anti-apoptotic viral protein, BHRF1. Therefore, m6A shows potential as a target of lytic induction therapy for EBV-associated cancers, including Burkitt lymphoma.

Epstein–Barr virus (EBV), which encodes >80 genes and nearly 50 non-coding RNAs, is a double-stranded DNA virus. EBV is associated with various types of lymphomas and lymphoproliferative disorders not only of B-cell but also T/NK-cell origin. However, the oncogenic mechanism remains poorly understood, including the EBV receptors expressed on T/NK cells, relationship of EBV with host genes, and epigenetic regulation of EBV and host genes. The roles of host and viral non-coding RNAs during tumorigenesis have been elucidated. EBV encodes at least 49 mature microRNAs (miRNAs), of which 44 are located in BamHI-A rightward transcripts (BARTs) region, and the remaining five are located in BamHI-H rightward fragment 1. BART miRNAs modulate cell differentiation, proliferation, apoptosis, and the cell cycle, and they are considered positive regulators of oncogenesis. We and others have recently reported that EBV-positive lymphomas frequently possess large deletions in BART miRNA clusters, suggesting that some viral miRNAs have suppressive effects on oncogenesis, and that deletion of these miRNAs may aid lymphoma formation.

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and antiviral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, we infected EBV-negative Akata cells with EBV-EGFP and performed transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprogramming of cell growth by EBV, and a burst of viral lytic gene expression in the early phase of infection. Experimental and mathematical investigations demonstrate that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Fate mapping using recombinant EBV provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency. Furthermore, we find that the BZLF1 protein, which is a key regulator of reactivation, was dispensable for abortive lytic infection in the pre-latent phase, suggesting the divergent regulation of viral gene expressions from a productive lytic infection.

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

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. [ABSTRACT FROM AUTHOR]

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-κB), 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-κB, 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. [ABSTRACT FROM AUTHOR]