Abstract
ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A), at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E), Z(R187K) and Z(K188A), were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1), the single-stranded DNA-binding protein (BALF2) and the DNA polymerase processivity factor (BMRF1), partially rescued the replication defect in these mutants and enhanced ZEBRA's interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.
Highlights
There are many gaps in our understanding of the process by which the Epstein-Barr virus (EBV) lytic replication machinery assemble on DNA sites present in the viral genome.EBV encodes an essential bZIP protein known as ZEBRA that functions as a transcription activator of viral and cellular genes and as an origin binding protein during lytic DNA replication
ZEBRA interacts both with promoters and with origins of lytic replication through DNA sequences known as ZEBRA response elements (ZREs) that are common to both types of DNA regulatory regions [2,3,4]
We studied five ZEBRA mutants that are impaired in activation of replication but are wild-type in the capacity to induce transcription of early viral genes
Summary
EBV encodes an essential bZIP protein known as ZEBRA (aka Zta, Z and BZLF1) that functions as a transcription activator of viral and cellular genes and as an origin binding protein during lytic DNA replication. ZEBRA interacts both with promoters and with origins of lytic replication through DNA sequences known as ZEBRA response elements (ZREs) that are common to both types of DNA regulatory regions [2,3,4]. It is unknown how ZEBRA distinguishes between a replication site and a transcription activation site. Since ZEBRA augments the histone acetyl transferase (HAT) activity of CBP, interaction of ZEBRA with CBP increases promoter accessibility [9]
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