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-aminoacid-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.

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

“…These included seven mutants, K178D, K178E, R179E, R183E, R190E, K194D, and K194E, in which a basic aa was replaced by an acidic aa. These mutants were selected for study because 11 of the 19 non-DNA-binding mutants generated by Heston et al (30) had replaced a basic aa with an acidic aa. The other three mutants were N182K in which a neutral aa was replaced by a basic aa, N182E in which a neutral aa was replaced by an acidic aa, and S186E in which the well studied serine at position 186, required for BRLF1 activation and absent from other AP-1-binding proteins (39), was changed to a glutamate.…”

“…DNA Replication Assays-DNA was extracted, purified, and digested with BamHI as described in Heston et al (30). After electrophoresis, DNA was denatured with 0.4 M NaOH and transferred to a Zeta-Probe GT genomic membrane (Bio-Rad) by Southern blotting procedure.…”

“…In immunoblot experiments, ZEBRA was detected using a rabbit polyclonal antibody described previously (21). Rta and BLRF2 protein were detected using rabbit polyclonal antisera described previously (30). BFRF3 was detected with a rabbit polyclonal antibody (S1931-1) raised against full-length BFRF3 protein expressed and purified from E. coli.…”

“…Gene-The procedure for construction of the bzlf1 mutants has been described (30). The 10 mutants analyzed in this study were as follows: K178D (AAG to GAC), K178E (AAG to GAA), R179E (CGA to GAA), N182K (AAT to AAG), N182E (AAT to GAG), R183E (CGG to GAG), S186E (TCC to GAG), R190E (CGG to GAG), K194D (AAG to GAC), and K194E (AAG to GAA).…”

“…Heston et al (30) analyzed an extensive series of single amino acid substitutions introduced throughout the basic domain of ZEBRA. Of these mutants, 27 retained the capacity to bind DNA yet showed specific defects in the ability to activate different phases of the lytic cycle.…”