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.

FIG. 1

Capacity of Z(S186) mutants to activate the EBV BRLF1 gene. Raji cells were chemically induced (lane 1), untreated (lane 2), or transfected with various amounts of expression vector (from 1.2 to 14.3 μg) plus pHD1013 to a total of 15 μg of plasmid DNA (lanes 3 to 9) to ensure that the expression level of each of the mutants equaled or exceeded the wild-type ZEBRA expression level. RNA and protein extracts were prepared 24 h after transfection. Each lane of a Northern blot (A) contained RNA from 2 × 10⁶ cells. The Northern blot was probed with BRLF1 and the H1 component of RNase P to control for RNA loading. Each lane of an immunoblot (B) contained an extract of 3 × 10⁶ cells. The immunoblot was probed with monospecific antibodies to Rta, to ZEBRA, and to β-actin to control for protein loading. T/B, tetradecanoylphorbol acetate/n-butyrate.

FIG. 2

Comparison of DNA binding by ZEBRA and Z(S186A) by competition EMSA. The oligonucleotide probe contained an AP-1 heptamer site (A) or the ZRE-R site (B). In panel A, 1 U of ZEBRA and Z(S186A) was determined to be that amount of bacterial extract that contained an approximately equivalent amount of immunoreactive ZEBRA or mutant protein; in panel B, the volume of cell extract required to achieve equivalent immunoreactivity is indicated. The binding of ZEBRA and Z(S186A) was assessed alone or in the presence of increasing amounts of competitor ZΔ131 extract. The proportion of probe that was bound by each protein was quantitated by phosphorimage analysis and is indicated below the gel.

FIG. 3

Comparison of DNA binding activities by ZEBRA and a group of Z(S186) mutant proteins. (A) Bacterial lysates containing ZEBRA or Z(S186) mutant proteins were examined by immunoblotting with rabbit antiserum to ZEBRA. The level of expressed protein was quantitated by phosphorimagery and adjusted so that all DNA binding reactions contained approximately equal amounts of immunoreactive protein. (B) EMSAs of ZEBRA and Z(S186) mutant proteins with duplex oligonucleotide probes harboring one of several known ZEBRA binding sites labeled with polynucleotide kinase. Lane 6 contained extract of bacterial cells transformed with the vector pET-11d; lane 7 contained extract of E. coli BL21(DE3) cells alone. Only the portion of the gel containing probe that was shifted by the protein is shown.

FIG. 4

Transcriptional activation of promoter/CAT reporters by ZEBRA and a panel of Z(S186) mutants. (A) Activation of BMRF1p/CAT. BJAB cells were transfected with 10 μg of BMRF1 promoter reporter plasmid and 10 μg of wild-type (wt) or mutant BZLF1 expression plasmid. The wild type and mutants are indicated by the amino acid present at position 186. The data represent the mean and standard error of the mean of three separate transfections. Fold activation is percent acetylation in the presence of BZLF1 or mutant expression vector/percent acetylation in the presence of CMV vector alone. (B) Activation of BRLF1p/CAT (Rp/CAT). BJAB cells were cotransfected with 10 μg of Rp/CAT reporter and with a total of 10 μg of activator plasmid. The activator consisted of 5 μg of BZLF1 or mutant expression vector plus 5 μg of vector alone (CMV) (solid bars) or 5 μg of Rta (open bars). Transfection efficiency was corrected by transfection of 1 μg of pGL2 Basic + HMP. Fold activation is relative to the level with reporter together with 10 μg of CMV. Synergy index: percent acetylation by ZEBRA or Z(S186) mutant in the presence of Rta/percent acetylation by ZEBRA or mutant alone plus percent acetylation by Rta alone.

FIG. 5

Capacity of Rta to rescue BMRF1 expression following cotransfection with various Z(S186) mutants. Raji cells were chemically treated (lane 1) or transfected with 10 μg of pRTS, the expression vector for Rta (lanes 2 and 9), or with wild-type or Z(S186) mutant expression plasmids (lanes 3 to 8 and 10 to 15). In lanes 2 to 10, the cells received an additional 10 μg of empty vector pRTS; in lanes 9 to 15, the cells received pRTS/Rta. For the Northern blot shown in panel A, cells were harvested 46 h after transfection; for the immunoblot in panel B, cells were harvested 48 h after transfection. T/B, tetradecanoylphorbol acetate/n-butyrate.

FIG. 6

Summary of the behavior of the panel of Z(S186) mutants. (A) Hypothetical model for behavior of ZEBRA mutants on Rp. ZEBRA mutants that are unable to activate Rp have two possible defects: a low affinity of binding to Rp and an inability to be phosphorylated. (B) Hypothetical model for capacity of ZEBRA mutants to synergize with Rta on the BMRF1 promoter. Synergy requires mutual contact between Rta, ZEBRA, and protein X. ZEBRA mutants that do not synergize with Rta are unable to contact protein X.