Functional and physical interactions between the Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1: Effects on EBV transcription and lytic replication

Abstract

The Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1 are both essential for lytic EBV replication. BZLF1 is a transcriptional activator which binds directly to the lytic origin of replication (oriLyt) and plays a critical role in the disruption of viral latency. The BMRF1 protein is required for viral polymerase processivity. Here we demonstrate that the BMRF1 gene product functions as a transcriptional activator and has direct (as well as indirect) interactions with the BZLF1 gene product. The BMRF1 gene product activates an essential oriLyt promoter, BHLF1, but does not activate two other early EBV promoters (BMRF1 and BHRF1). Direct interaction between the BMRF1 and BZLF1 gene products requires the first 45 amino acids of BMRF1 and the bZip domain of BZLF1. The effect of the BZLF1-BMRF1 interaction on early EBV transcription is complex and is promoter specific. The oriLyt BHLF1 promoter is activated by either the BZLF1 or BMRF1 gene product alone and is further activated by the combination of the BZLF1 and BMRF1 gene products. Enhanced activation of BHLF1 transcription by the BMRF1-BZLF1 combination does not require direct interaction between these proteins. In contrast, BZLF1-induced activation of the BMRF1 promoter is inhibited in the presence of the BMRF1 gene product. A point mutation in the BZLF1 protein (amino acid 200), which prevents in vitro interaction with the BMRF1 protein but which does not reduce BZLF1 transactivator function, allows the BZLF1 protein to activate the BMRF1 promoter equally well in the presence or absence of the BMRF1 gene product. Therefore, direct interaction between the BZLF1 and BMRF1 proteins may inhibit BZLF1-induced transcription of the BMRF1 promoter. BZLF1 mutated at amino acid 200 is as efficient as wild-type BZLF1 in promoting replication of an oriLyt plasmid. However, this mutation reduces the ability of BZLF1 to induce lytic replication of the endogenous viral genome in D98/HE-R-1 cells. Our results indicate that functional and physical interactions between the BMRF1 and BZLF1 proteins may modulate the efficiency of lytic EBV infection. The BMRF1 gene product clearly has a transcriptional, as well as replicative, role during lytic EBV infection.

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

“…The viral transcription factor BZLF1, which is indispensable for DNA replication, has been shown to interact with components of the EBV helicase–primase complex (Gao et al ., 1998) as well as with BMRF1, the accessory factor of the viral DNA polymerase BALF5 (Zhang et al ., 1996). These interactions are likely to contribute to DNA replication at the upstream component of oriLyt .…”

“…These interactions are likely to contribute to DNA replication at the upstream component of oriLyt . We were interested to know if similar interactions occur at the downstream component since BMRF1 has been proposed to transcriptionally activate the downstream component of oriLyt (Zhang et al ., 1996). Although BMRF1 does not bind specifically to the downstream component, BMRF1 could be tethered indirectly to the downstream component by factors which do bind to oriLyt (Zhang 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). The single‐stranded DNA‐binding protein BALF2 may in turn contact the helicase–primase subcomplex (Gao et al ., 1998).…”

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

“…The ease with which ZEBRA's activation region can be exchanged in the B-lymphocyte system may be related to the unique properties of the ZEBRA bZIP-like region. In addition to mediating dimerization and DNA recognition, the bZIP-like region in ZEBRA has been found to be essential for binding to the cellular proteins p53 and NF-B/p65 and to the viral BMRF1 (EA-D) protein (21,46,47). The bZIP region has also been recently demonstrated to mediate growth arrest at the G0/G1 phase in epithelial cells (6,8).…”

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

“…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). The single-stranded DNA-binding protein BALF2 may in turn contact the helicase-primase subcomplex (Gao et al, 1998).…”