Expression and function of ZEBRA

Naomi Taylor, Yale University.

This is an Open Access Thesis


Epstein-Barr virus (EBV) encodes a protein, ZEBRA, which enables the virus to switch from a latent to a lytic life cycle. ZEBRA expression was studied in lymphoid cells harboring either standard virus or a mixture of standard and defective viruses. ZEBRA protein could not be detected in cells latently infected with standard EBV but was constitutively expressed in cells containing both defective and standard EBV genomes. Experiments indicated that regulation of transcription of the ZEBRA gene (BZLF1) is a pivotal event in the control of EBV replication. ZEBRA specific BZLF1 transcripts were spontaneously expressed in cells harboring defective virus but were only synthesized by the latent virus after chemical induction. BZLF1 transcription was sensitive to inhibition of protein synthesis but not to inhibition of lytic viral DNA synthesis. This suggests that ZEBRA is an early replicative protein whose expression is dependent on the synthesis of a viral or cellular factor.The basis domain of the ZEBRA protein is homologous to the Fos/Jun oncogene family and shares their ability to bind the canonical AP-1 site (TGAGTCA). However, ZEBRA does not contain a leucine zipper domain; a motif necessary for DNA binding of Fos/Jun proteins. Additionally, ZEBRA binds to sites which deviate from the AP-1 consensus sequence. Thus, it was of interest to study the DNA binding properties of the ZEBRA protein.Deletional mutagenesis of the BZLF1 cDNA indicated that amino acids 172-227, representing the basic and putative dimerization domains, were required for specific binding to AP-1 and divergent sites. Mutagenesis of three basic amino acids, which are conserved in Fos, abrogated ZEBRA binding to all target sequences. Additionally, ZEBRA was determined to bind DNA as a homodimer.DNA binding studies of ZEBRA and a Fos-GCN4 chimera indicated that although these proteins have homology in their basic DNA binding domains, they have different cognate binding specificities. The autoregulated BZLF1 promoter contains three divergent AP-1 sequences; Fos-GCN4 uniquely recognized one of the sites while ZEBRA bound only the other two sequences. Additionally, ZEBRA, but not Fos, was found to be phosphorylated by casein kinase II in-vitro. This phosphorylation abrogated ZEBRA binding to all of its target DNA sequences. Regulation of ZEBRA's DNA binding activity may be required for the progression of the EBV replicative cycle.