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Background: Epstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt's lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. The current approach for EBVassociated lymphoma involves chemotherapy to eradicate cancer cells, however, normal cells may be injured and organ dysfunction may occur with currently employed regimens. This research is focused on employing arsenic trioxide (ATO) as EBV-specific cancer therapy takes advantage of the fact the EBV resides within the malignant cells. Methods and results: Our research reveals that low ATO inhibits EBV gene expression and genome replication. EBV spontaneous reactivation starts as early as 6 h after re-suspending EBV-positive Mutu cells in RPMI media in the absence of ATO, however this does not occur in Mutu cells cultured with ATO. ATO's inhibition of EBV spontaneous reactivation is dose dependent. The expression of the EBV immediate early gene Zta and early gene BMRF1 is blocked with low concentrations of ATO (0.5 nM - 2 nM) in EBV latency type I cells and EBV-infected PBMC cells. The combination of ATO and ganciclovir further diminishes EBV gene expression. ATO-mediated reduction of EBV gene expression can be rescued by co-treatment with the proteasome inhibitor MG132, indicating that ATO promotes ubiquitin conjugation and proteasomal degradation of EBV genes. Co-immunoprecipitation assays with antibodies against Zta pulls down more ubiquitin in ATO treated cell lysates. Furthermore, MG132 reverses the inhibitory effect of ATO on anti-IgM-, PMA- and TGF-β-mediated EBV reactivation. Thus, mechanistically ATO's inhibition of EBV gene expression occurs via the ubiquitin pathway. Moreover, ATO treatment results in increased cell death in EBV-positive cells compared to EBV-negative cells, as demonstrated by both MTT and trypan blue assays. ATO-induced cell death in EBV-positive cells is dose dependent. ATO and ganciclovir in combination further enhances cell death specifically in EBV-positive cells. Conclusion: ATO-mediated inhibition of EBV lytic gene expression results in cell death selectively in EBV-positive lymphocytes, suggesting that ATO may potentially serve as a drug to treat EBV-related lymphomas in the clinical setting. [ABSTRACT FROM AUTHOR]

Abstract: The Epstein Barr virus (EBV) plays a role in maintenance of the tumor phenotype in a number of human cancers. The EBV latency replication factor, EBNA1, is required for persistence of the EBV episome, is anti-apoptotic, and is universally expressed in all EBV-associated tumors. Here, we show that EBNA1-specific siRNAs can inhibit EBNA1 expression and function. siRNAs were generated against three target sites in the EBNA1 messenger RNA, and two of these were found to inhibit EBNA1 expression from an ectopic EBNA1 expression cassette. EBNA1 siRNAs also inhibit endogenously expressed EBNA1 in EBV-positive epithelial and B-cell lines. Using a mini-EBV replication model, siRNA-mediated inhibition of EBNA1 expression suppressed the episomal maintenance function of EBNA1. Lastly, introduction of an EBNA1 siRNA into an EBV-positive tumor cell line inhibited tumor cell growth/survival. These data suggest that siRNAs against EBNA1 may have therapeutic value in EBV-associated diseases. [Copyright &y& Elsevier]

Previous investigations proposed a link between the Epstein-Barr virus (EBV) and lung cancer (LC), but the results are highly controversial largely due to the insufficient sample size and the inherent limitation of the traditional viral screening methods such as PCR. Unlike PCR, current next-generation sequencing (NGS) utilizes an unbiased method for the global assessment of all exogenous agents within a cancer sample with high sensitivity and specificity. In our current study, we aim to resolve this long-standing controversy by utilizing our unbiased NGS-based informatics approaches in conjunction with traditional molecular methods to investigate the role of EBV in a total of 1127 LC. In situ hybridization analysis of 110 LC and 10 normal lung samples detected EBV transcripts in 3 LC samples. Comprehensive virome analyses of RNA sequencing (RNA-seq) data sets from 1017 LC and 110 paired adjacent normal lung specimens revealed EBV transcripts in three lung squamous cell carcinoma and one lung adenocarcinoma samples. In the sample with the highest EBV coverage, transcripts from the BamHI A region accounted for the majority of EBV reads. Expression of EBNA-1, LMP-1 and LMP-2 was observed. A number of viral circular RNA candidates were also detected. Thus, we for the first time revealed a type II latency-like viral transcriptome in the setting of LC in vivo. The high-level expression of viral BamHI A transcripts in LC suggests a functional role of these transcripts, likely as long non-coding RNA. Analyses of cellular gene expression and stained tissue sections indicated an increased immune cell infiltration in the sample expressing high levels of EBV transcripts compared to samples expressing low EBV transcripts. Increased level of immune checkpoint blockade factors was also detected in the sample with higher levels of EBV transcripts, indicating an induced immune tolerance. Lastly, inhibition of immune pathways and activation of oncogenic pathways were detected in the sample with high EBV transcripts compared to the EBV-low LC indicating the direct regulation of cancer pathways by EBV. Taken together, our data support the notion that EBV likely plays a pathological role in a subset of LC. [ABSTRACT FROM AUTHOR]

Transcription of the immediate early gene BZLF1 is mediated initially through the activation of cellular transcription factors. Reporter-based studies have provided evidence that following this initial activation, the BZLF1 gene product Zta may be involved in an autoactivation loop through binding to its promoter Zp. In contrast, other reports have shown that transfection of a Zta expression vector in latently infected cells does not activate endogenous Zp. Using chromatin immunoprecipitation (ChIP) assays, we show here that Zta binds to endogenous Zp following induction of the lytic cycle by anti-Ig and TGF-beta1 and that binding occurs early enough to play a role in the activation of Zp. We have also generated a dominant-negative Zta and shown that it inhibits activation of endogenous Zp. These data support a two-step model for Zp activation during reactivation involving initial activation by cellular factors followed by an autoactivation step. [Copyright &y& Elsevier]

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication. [ABSTRACT FROM AUTHOR]

The relationship between Epstein Barr Virus (EBV) and miR-155 is well established. EBV infection induces miR-155 expression, which is expressed at higher levels in EBV latency type III cells compared to EBV latency type I cells. However, the mechanism by which EBV latency genes activate miR-155 expression is still unclear. Here we present data showing that DNA methylation regulates miR-155 expression. We also provide evidence that the AP1 signaling pathway is involved in EBV-mediated miR-155 activation, and that Bay11 influences signaling of the miR-155 promoter AP1 element. Lastly, we show that LMP2A, LMP1 and EBNAs cannot activate miR-155 expression alone, indicating that the regulation of miR-155 by EBV is dependent on more than one EBV gene or cell signaling pathway. We conclude that the regulation of miR-155 in EBV-positive cells occurs through multiple cell signaling processes involving EBV-mediated chromatin remodeling, cell signaling regulation and transcription factor activation. [ABSTRACT FROM AUTHOR]