Regulation of the Latent-to-Lytic Switch of Epstein-Barr Virus by TGF-beta and Ikaros

Epstein-Barr Virus (EBV) establishes latent infection in B-cells, occasionally reactivating into lytic replication in the presence of lytic-inducing stimuli, e.g., TGF-beta, which stimulate expression of EBV's latent-lytic switch BZLF1 gene. Here, I investigated the mechanism by which TGF-beta activates transcription from the BZLF1 promoter, Zp. I identified four additional Smad-binding elements on Zp, named SBE2 to SBE5. Each SBE contributed to Smad-mediated activation of Zp, with SBE2 and SBE4 being of greatest importance; together with SBE1, they accounted for essentially all of the activation. SBE2 overlaps the ZEB-binding ZV element of Zp. Depending upon post-translational modifications, Smad4 competed with ZEB1 for binding or formed a complex with ZEB1 on the ZV element in vitro. In transiently transfected cells, ZEB1 inhibited transcriptional activation from Zp by activated Smads. Thus, I conclude that TGF-beta induces EBV lytic reactivation by activating BZLF1 gene expression through multiple SBEs acting in concert.;Ikaros is a DNA-binding zinc-finger protein that is critical for lymphocyte development and functions as a tumor suppressor in acute lymphoblastic leukemia. Ikaros can either activate or repress transcription of target genes via chromatin remodeling. Here, I demonstrated that full-length Ikaros (IK-1) acted as a critical factor in maintenance of viral latency in some EBV-positive Burkitt's lymphoma cell lines. Both knockdown of Ikaros expression by shRNAs or ectopic expression of a dominant-negative isoform (IK-6) induced lytic replication. The mechanism by which Ikaros promoted EBV latency did not involve direct binding to the EBV immediate-early BZLF1 or BRLF1 gene. Rather, Ikaros regulated expression of cellular Oct-2 and Bcl-6, inhibiting EBV reactivation and plasma cell differentiation, respectively. IK-1 strongly interacted and co-localized with the EBV R protein within cells. The residues critical for IK/R interaction mapped within the DNA-binding domain of R and C-terminal domain of Ikaros. Presence of R alleviated transcriptional repression mediated by IK-1, with IK-1 synergizing with Z and R to increase EBV lytic-gene expression. Therefore, I conclude that Ikaros plays two distinct roles at different stages of EBV's life cycle: it contributes to maintaining viral latency by indirect mechanisms; and it synergizes with Z and R to enhance lytic replication.