Multivalent sequence recognition by Epstein-Barr virus Zta requires cysteine 171 and an extension of the canonical B-ZIP domain.

Epstein-Barr virus (EBV) immediate-early protein Zta is a member of the basic-leucine zipper (B-ZIP) family of DNA binding proteins that has an unusual capacity to recognize multiple DNA recognition sites, including AP-1 and C/EBP binding sites. To better understand the structure and function of Zta, we have mutagenized cysteine residues within or adjacent to the B-ZIP domain. We found that serine substitution for cysteine 171 (C171S), which lies outside and amino terminal to the B-ZIP basic region, completely abrogates Zta capacity to initiate lytic cycle replication. C171S disrupted Zta transcription activation function of several EBV lytic cycle promoters, including the BMRF1 gene (EA-D) and the other lytic activator, Rta. Overexpression of Rta could not rescue the C171S defect for transcription reactivation or viral DNA replication. Zta C171S was defective for binding to these promoters in vivo, as measured by chromatin immunoprecipitation assay. Purified Zta C171S bound AP-1 sites similar to wild-type Zta, but it was incapable of binding several degenerate Zta sites, including a consensus C/EBP site. Zta truncation mutations reveal that residues N terminal to the B-ZIP (amino acids 156 to 178) confer C/EBP binding capacity to the otherwise AP-1-restricted DNA recognition function. Comparison among viral orthologues of Zta suggest that a conserved N-terminal extension of the consensus B-ZIP domain is required for this multivalent DNA recognition capacity of Zta and is essential for viral reactivation.