Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation

Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication requ ire the transcriptional activation function of the viral immediate-early pr otein Zta. We describe a series of alanine substitution mutations in the Zt a activation domain that reveal two functional motifs based on amino acid c omposition. Alanine substitution of single or paired hydrophobic aromatic a mino acid residues resulted in modest transcription activation defects, whi le combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid res idue E27, D35, or E54 caused severe transcription defects on, most viral pr omoters. Promoter- and cell-specific defects were observed for some substit ution mutants. Aromatic residues were required for Zta interaction with TFI IA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP hist one acetyltransferase activity in vitro. In contrast, acidic amino acid sub stitution mutants interacted with TFIIA-TFIID and CBP indistinguishably fro m the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed b y most Zta mutants, but acidic residue mutations led to reduced, while arom atic substitution mutants led to increased SP100 nuclear staining. Acidic r esidue substitution mutants had more pronounced defects in transcription ac tivation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant , K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates vi ral reactivation through two nonredundant structural motifs, one of which i nteracts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription .