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

Abstract

The ZEBRA protein mediates the switch between the latent and lytic life cycles 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, 1997). We now describe three distinct phenotypes of ZEBRA mutants bearing different amino acid substitutions at S186. These phenotypes are based on the capacity of the mutants to activate expression of the BRLF1 and BMRF1 genes, which are targets of ZEBRA’s action, and to synergize with the BRLF1 gene product Rta (R transactivator) in activating expression of downstream genes. 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 activate early lytic cycle products from the latent virus, and not rescued by overexpression of Rta. A third class, Z(S186A), although unable by itself to activate BRLF1 or other lytic cycle genes, synergized with Rta. Rta rescued the capacity of Z(S186A) to activate the BMRF1 early lytic cycle gene from the latent virus. All mutant classes bound to DNA in vitro, although their capacity 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 synergize with Rta, synergy with Rta does not appear to be dependent on phosphorylation of S186. S186 likely mediates DNA recognition on the BRLF1 promoter in the context of the latent virus, protein-protein interactions, or both. The Z(S186) mutants define the amino acid side chains required for these functions.

Sequence analysis of EBV immediate-early gene BZLF1 and BRLF1 in lymphomas

“…The distribution of mutations at residue 68 and 76 which were located in the most important subregion for transactivation [Lieberman and Berk, ; Packham et al, ; Lieberman and Berk, ; Flemington et al, ] in lymphomas was different from the other two tumors (EBVaGCs and nasopharyngeal carcinomas) or throat washing samples from healthy donors. In the DNA binding domain AA 186 plays an important role in mediating DNA recognition on the BRLF1 promoter in the context of the latent virus [Francis et al, ]; AA 189 may influence the DNA‐binding efficiency of ZEBRA [Schelcher et al, ]. In this study AAs 186 and 189 were found to be conserved, further verifying their important biological roles.…”

Demonstration of Epstein–Barr virus in odontogenic and nonodontogenic tumors by the polymerase chain reaction (PCR)

“…Additionally, following primary infection involving the salivary gland, EBV remains latent in epithelial cells of the normal salivary gland and periodically becomes reactivated (18). EBV is actively being shed into saliva as free cells and the detection of EBV by PCR in saliva from lymphoproliferative disorders can be a predictor of disease activation (19).…”

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation

“…Previous work demonstrated that ZEBRA's serine 186 (S186) is the major contributor to recognition of methylated DNA (12). Upon mutation of this residue to alanine, ZEBRA recognition of methylated DNA is ablated, essentially eliminating its function in activation of the EBV lytic cascade (24). Several crystal structures have attempted to explain ZEBRA's selectivity for methylated DNA.…”