We previously showed that the cellular proteins ZEB1 and ZEB2/SIP1 both play key roles in regulating the latent-lytic switch of Epstein-Barr Virus (EBV) by repressing BZLF1 gene expression. We investigated here the effects of cellular microRNA (miRNA) 200 (miR200) family members on the EBV infection status of cells. We show that miR200b and miR429, but not miR200a, can induce EBV-positive cells into lytic replication by downregulating expression of ZEB1 and ZEB2, leading to production of infectious virus. The levels of miR200 family members in EBV-infected cells strongly negatively correlated with the levels of the ZEBs (e.g., ؊0.89 Reactivation of EBV out of latency into lytic replication is necessary for the viral progeny to pass from host to host. It occurs naturally in infected individuals at a low frequency; periodic shedding of the virus into saliva allows for transmission (75). It remains unclear how reactivation occurs in vivo. The product of the BZLF1 gene, known as BZLF1 (also called ZEBRA, Z, Zta, and EB1), is a key player in switching EBV from latency into lytic replication in cells in culture (16,17,25,80; reviewed in references 74 and 75).BZLF1 is a multifunctional DNA-binding protein belonging to the bZIP member of transcription factors (12). By activating transcription of other viral genes and binding to the viral origin of lytic replication, oriLyt, BZLF1 can induce viral genome replication and expression of a cascade of other EBV lytic genes necessary for packaging of the genome into virion particles and exit from the host cell (40,52,74,76,77). BZLF1 can also interact with several cellular proteins, affecting their activities and cellular localization, further contributing to viral reactivation (61, 74). Because of BZLF1's central role in reactivation of EBV out of latency into lytic replication, factors that regulate its expression have been implicated as potential therapeutics for treating patients with EBV-associated malignancies.Our laboratory previously reported that the cellular proteins ZEB1 (also known as ␦EF1, TCF8, AREB6, ZFHEP, NIL-2A, ZFHX1A, and BZP) and ZEB2 (also known as SIP1, SMA-DIP1, ZFHX1B, and KIAA0569) can both bind to a sequence element, termed ZV, located within the BZLF1 promoter, Zp (22,47,48,92); a second element, ZVЈ, synergizes with the ZV element for higher-affinity binding of the ZEBs to Zp via their two zinc fingers (Fig. 1A) (X. Yu, P. McCarthy, D. Gorlen, and J. E. Mertz, unpublished data). Both of these ZEB family members bind to target DNA via E-box-binding sequences resembling 73). Depending on interactions with coactivators and corepressors and associations with histone deacetylases (HDACs) (68,71,84), ZEB1 can either activate or repress transcription of its target genes (68,70,72); to date, ZEB2 has been reported only to repress transcription (67). We showed that exogenous expression of either ZEB1 or ZEB2 leads to repression of transcription driven from Zp in transient transfection assays (22,48,92). Mutation of the ZV element in the context of the B9...

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV’s natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies.

Transforming growth factor ␤ (TGF-␤) physiologically induces Epstein-Barr virus (EBV) lytic infection by

Purpose Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin’s lymphoma (HL) and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein-Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection. Experimental Design We studied the ability of LTP to reactivate latently infected EBV-positive cell lines in vitro and in vivo, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide and dexamethasone (LTD) combination. Results Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene BZLF1, the early gene BMRF1, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide>lenalidomide>thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt’s lymphoma cells in vitro and in vivo. EBV reactivation was related to phosphatidylinositol-3 kinase (PI3K) stimulation and Ikaros suppression, and blocked by the PI3K-δ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared to lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation Conclusions We conclude LTP may reactivate EBV-positive resting memory B-cells thereby enhancing EBV lytic cycle and host immune suppression.