Activation domain requirements for disruption of Epstein-Barr virus latency by ZEBRA

Abstract

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) can be disrupted by expression of the EBV ZEBRA protein. ZEBRA, a transcriptional activator, initiates the EBV lytic cascade by activating viral geneexpression. ZEBRA is also indispensable for viral replication and binds directly to the EBV lytic origin of replication. The studies described herein demonstrate that the activation domain of ZEBRA is not unique and can be replaced by a heterologous acidic, proline-rich, or glutamine-rich activation domain. ZEBRA activation domain swap constructs retain ZEBRA's native abilities to activate specific EBV promoters, to disrupt EBV latency, and to stimulate replication at the EBV lytic origin. Additional work, employing sequential and internal deletions of ZEBRA's N-terminal activation domain, indicates that its separate activities are not attributable to specific subdomains but are spread throughout its N terminus and therefore cannot be inactivated by deleting localized regions. MATERIALS AND METHODSCells. The B-cell lymphoma lines Clone 16 (CL16), Raji, BL41/CL16, and BJAB were maintained in RPMI medium supplemented with 8% fetal calf serum and antibiotics at 37°C under a 5% CO 2 atmosphere (4). CL16 cells are human B lymphocytes which harbor the nontransforming P3HR-1 strain of EBV in the latent state (36). Raji cells are human B lymphocytes which contain a deleted EBV episome that can be induced to express early antigen, but which cannot replicate (24). The BL41/CL16 cell line is a derivative of the EBV-negative Burkitt's lymphoma BL41 cell line, which has been stably infected with the EBV CL16 virus. After prolonged culture in vitro, the EBV in BL41/CL16 cells has become progressively more tightly latent and can no longer be induced by * Corresponding

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

“…assay (58). However, in studies in other laboratories using different assay systems for oriLyt replication, replacement or mutation of the Zta activation domain had not been able to uncover a replication specific contribution for this domain (1,60). While the reason for the different results cannot be ascribed with certainty, the present study allows speculation.…”

“…A transcriptional contribution to replication by Zta seems likely. Removal of the Zta transcriptional activation domain abolishes replication activity (1,58,60), and fusion of an additional heterologous activation domain increases reactivation efficiency (2). As a transcription factor, Zta may contribute either by disrupting nucleosome formation and increasing the accessibility of the origin to the replication complex or by introducing topological changes in the DNA that facilitate replication initiation (28,37).…”

“…Zta has a second role in lytic cycle reactivation, serving as an essential regulatory protein for replication of the lytic origin, oriLyt (1,21,58,61). EBV oriLyt is duplicated with one copy located within the BamHI H fragment and a second copy located in the region that is deleted in the sequenced B95-8 isolate (30,35).…”

Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt

“…In contrast, viral lytic proteins cannot be detected in the latent phase, but stimuli, such as TPA, lead to the activation of the lytic activator BZLF1, which in turn results in the induction of viral lytic genes including viral DNA replication proteins (reviewed in Speck et al ., 1997). BZLF1 is also capable of activating oriLyt directly by binding to DNA motifs, so‐called BZLF1‐responsive DNA elements (ZRE), located in the upstream component (Lieberman et al ., 1990; Schepers et al ., 1993a, 1996; Sarisky et al ., 1996; Askovic and Baumann, 1997). Protein–protein interactions have been demonstrated to exist between the DNA‐binding domain of BZLF1 and the DNA polymerase accessory factor (BMRF1) (Zhang et al ., 1996), and between the transactivation domain of BZLF1 and both the viral helicase (BBLF4) and the primase subcomplex (composed of BSLF1 and BBLF2/3) (Gao et al ., 1998).…”

Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

“…Numerous cellular and viral promoters contain binding In contrast, viral lytic proteins cannot be detected in the latent phase, but stimuli, such as TPA, lead to the activation of the lytic activator BZLF1, which in turn results in the induction of viral lytic genes including viral DNA replication proteins (reviewed in Speck et al, 1997). BZLF1 is also capable of activating oriLyt directly by binding to DNA motifs, socalled BZLF1-responsive DNA elements (ZRE), located in the upstream component (Lieberman et al, 1990;Schepers et al, 1993aSchepers et al, , 1996Sarisky et al, 1996;Askovic and Baumann, 1997). Proteinprotein interactions have been demonstrated to exist between the DNA-binding domain of BZLF1 and the DNA polymerase accessory factor (BMRF1) (Zhang et al, 1996), and between the transactivation domain of BZLF1 and both the viral helicase (BBLF4) and the primase subcomplex (composed of BSLF1 and BBLF2/3) (Gao et al, 1998).…”