Latency of Epstein-Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA

Abstract

ZEBReplication Activator (ZEBRA), a viral basic zipper protein that initiates the Epstein-Barr viral lytic cycle, binds to DNA and activates transcription through heptamer ZEBRA response elements (ZREs) related to AP-1 sites. A component of the biologic action of ZEBRA is attributable to binding methylated CpGs in ZREs present in the promoters of viral lytic cycle genes. Residue S186 of ZEBRA, Z(S186), which is absolutely required for disruption of latency, participates in the recognition of methylated DNA. We find that mutant cellular AP-1 proteins, Jun(A266S) and Fos(A151S), with alanine-to-serine substitutions homologous to Z(S186), exhibit altered DNA-binding affinity and preferentially bind methylated ZREs. These mutant AP-1 proteins acquire functions of ZEBRA; they activate expression of many viral early lytic cycle gene transcripts in cells harboring latent EBV but are selectively defective in activating expression of some viral proteins and are unable to promote viral DNA replication. Transcriptional activation by mutant c-Jun and c-Fos that have acquired the capacity to bind methylated CpG challenges the paradigm that DNA methylation represses gene expression.

Fig. 1.

Comparison of amino acid sequences in the basic domains of EBV ZEBRA and five cellular bZIP proteins. Boxed amino acids are identical or similar in all of the bZIP proteins. Dots indicate amino acids that contact bases in the crystal structure of ZEBRA (5) and the c-Fos/c-Jun heterodimer (12). Circled dots indicate amino acids that were mutated in the crystal structures. ZEBRA (S186) was mutated to alanine. The cysteines at positions ZEBRA (C189), c-Jun (C269), and c-Fos (C154) were all changed to serine. The unique serine 186 of ZEBRA is circled.

Fig. 2.

Point mutants in the basic domain of c-Jun and c-Fos substitute for ZEBRA in initiation of the EBV lytic cascade. (A) Plasmids encoding the indicated proteins were transfected in BZKO cells. The transfected cells were assessed for expression of EBV BRLF1, BMRF1, and BaRF1 mRNAs by Northern blotting. Relative activity was determined by densitometry of autoradiographs. (B) Jun(A266S) but not wt c-Jun activates expression of EBV Rta in individual cells. FLAG-tagged wt c-Jun (i–iii) or FLAG-tagged Jun(A266S) (iv–ix) were transfected into BZKO or 2089 cells that were fixed 43 h after transfection and incubated with primary antibodies to FLAG, Rta, and lamin B and appropriate secondary antibodies conjugated with FITC, DyLight 549, or DyLight 645. Images were obtained by confocal microscopy. (C) Heat map illustrating the relative capacity of wt and mutant forms of ZEBRA and cellular AP-1 proteins to activate expression of eight representative EBV lytic genes The primary data were generated from Northern blots presented in A and Fig. S2; the kinetic class and functions of these genes are described in Table S1.

Fig. 3.

Defect in the capacity of c-Jun(A266S) to activate expression of EBV early protein EA-D can be complemented by ZEBRA mutant Z(S186A). BZKO cells were transfected with plasmids encoding wt or mutant c-Jun(A266S) in the presence or absence of Z(S186A), a ZEBRA mutant that by itself is unable to activate the lytic cycle. The transfected cells were examined by Western blot for Rta, EA-D, and BGLF5 early proteins and BFRF3 late protein.

Fig. 4.

c-Jun(A266S) activates the EBV lytic cycle in a cell line derived from BL. HH514-16 cells were nucleofected with plasmids encoding wt ZEBRA, Rta, wt Jun, or the basic domain mutants c-Jun(A266S) or c-Fos(A151S). The cells were assessed for expression of BRLF1 and BZLF1 mRNAs (A) and BMRF1, BaRF1, and BFRF3 mRNAs (B).

Fig. 5.

Comparative binding of wt ZEBRA, c-Fos, and c-Jun and basic domain mutants Z(S186A), Fos(A151S), and Jun(A266S) to unmethylated and methylated ZEBRA response elements. Cell extracts of BZKO cells transfected with the indicated expression plasmids were prepared for EMSA. The probes were derived from sequences in the promoter of the BRFL1 gene (Rp) or the promoter of the BMLF1 gene (Table S2). These were Rp ZRE-1 (A), unmethylated Rp ZRE-2 (B), methylated Rp ZRE-2 (C), methylated ZRE-3 (D), and BMLF1p AP-1 (E). Relative activity was determined by densitometry of autoradiographs. (F) Immunoblot with antibody to FLAG in extracts prepared for the EMSA reactions.