title: A subset of replication proteins enhances origin recognition and lytic replication by the Epstein-Barr virus ZEBRA protein.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA.
authors.forename: Ayman
authors.initials: A
authors.lastname: El-Guindy
authors.forename: Lee
authors.initials: L
authors.lastname: Heston
authors.forename: George
authors.initials: G
authors.lastname: Miller
mesh_terms: D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D015534:Trans-Activators; D014764:Viral Proteins
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