Amino acid substitutions reveal distinct functions of serine 186 of the ZEBRA protein in activation of early lytic cycle genes and synergy with the Epstein-Barr virus R transactivator

The ZEBRA protein mediates the switch between the latent and lytic life cyc les of Epstein-Barr virus. Z(S186A), a point mutant in ZEBRA's basic domain in which serine 186 is changed to alanine, is unable to induce expression of lytic cycle mRNAs or proteins from the latent EBV genome even though it retains the ability to activate transcription from reporters bearing known ZEBRA-responsive promoters (A. L. Francis et al., J. Virol. 71:3054-3061, 1 997). We now describe three distinct phenotypes of ZEBRA mutants bearing di fferent amino acid substitutions at S186. These phenotypes are based on the capacity of the mutants to activate expression of the BRLF1 and BMRF1 gene s, which are targets of ZEBRA's action, and to synergize with the BRLF1 gen e product Rta (R transactivator) in activating expression of downstream gen es. One mutant class, represented by Z(S186T), was similar to the wild type , although reduced in the capacity to activate BRLF1 and BMRF1 early lytic cycle genes from the latent virus. A second class, represented by Z(S186C) and Z(S186G), was impaired in transcriptional activation, unable to activat e early lytic cycle products from the latent virus, and not rescued by over expression of Rta. A third class, Z(S186A), although unable by itself to ac tivate BRLF1 or other lytic cycle genes, synergized with Rta. Rta rescued t he capacity of Z(S186A) to activate the BMRF1 early lytic cycle gene from t he latent virus. All mutant classes bound to DNA in vitro, although their c apacity to bind to different ZEBRA response elements varied. Serine 186 of ZEBRA is a critical residue that is required for the distinct activities of induction of BRLF1 expression and for synergy with Rta. Since only Z(S186T ) among the mutants behaved similarly to the wild type, activation of BRLF1 likely requires phosphorylation of S186. However, since Z(S186A) could syn ergize with Rta, synergy with Rta does not appear to be dependent on phosph orylation of S186. S186 likely mediates DNA recognition on the BRLF1 promot er in the context of the latent virus, protein-protein interactions, or bot h. The Z(S186) mutants define the amino acid side chains required for these functions.