title: Amino acids in the basic domain of Epstein-Barr virus ZEBRA protein play distinct roles in DNA binding, activation of early lytic gene expression, and promotion of viral DNA replication.
abstract: The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.
authors.affiliation: Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Lee
authors.initials: L
authors.lastname: Heston
authors.forename: Ayman
authors.initials: A
authors.lastname: El-Guindy
authors.forename: Jill
authors.initials: J
authors.lastname: Countryman
authors.forename: Charles
authors.initials: C
authors.lastname: Dela Cruz
authors.forename: Henri-Jacques
authors.initials: HJ
authors.lastname: Delecluse
authors.forename: George
authors.initials: G
authors.lastname: Miller
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D000939:Epitopes; D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA