Evidence for coiled-coil dimer formation by an Epstein-Barr virus transactivator that lacks a heptad repeat of leucine residues.

Abstract

Two regions of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, share sequence homology with c-Fos, one of which corresponds to the DNA binding domain of c-Fos. ZEBRA does not, however, contain the heptad repeat of leucines present in the dimerization domains of leucine zipper proteins. Here it is shown that ZEBRA binds its recognition sites as a homodimer and that the region adjacent to the basic DNA binding domain is essential for dimerization. This region contains a 4-3 repeat of predominantly hydrophobic residues, which is precisely in register with the hydrophobic heptad repeat present in the leucine zipper proteins with respect to the basic DNA binding domain. A mutational analysis of ZEBRA supports a model for dimerization involving a coiled-coil interaction. These results indicate that a heptad repeat of leucines is not a structural requirement for formation of coiled-coil dimers by transcription factors.Epstein-Barr virus is a lymphotropic human herpesvirus that predominantly establishes a latent infection in B lymphocytes. Latent viral infection results in growth transformation of the infected lymphocytes, yielding continuously proliferating lymphoblastoid cells that are immortalized but not oncogenically transformed. Disruption of viral latency is determined by an intricate cascade of events initiating at the plasma membrane. In vitro, the viral lytic cycle can be triggered by a variety of reagents, including anti-immunoglobulin, calcium ionophore, butyrate, and the phorbol ester phorbol 12-myristate-13-acetate (1-4).Among the Epstein-Barr virus antigens known to be expressed during the onset of the viral lytic cycle, the BZLF1 gene product ZEBRA is unique in its ability to trigger the entire lytic cascade (5-7). ZEBRA shares two regions of homology with the cellular transactivator c-Fos, one of which is the DNA binding domain (8), and binds specifically to target sequences containing "AP-1 like" sites (8, 9, 11-13, *). However, the sequence similarity between ZEBRA and c-Fos does not extend into the leucine zipper dimerization domain of c-Fos adjacent to the basic DNA binding region (14)(15)(16). We have previously shown that mutations introduced into the basic region of ZEBRA abrogated DNA binding.* Furthermore, it has also been demonstrated that the region of ZEBRA adjacent to the basic domain is required for DNA binding (11, *), suggesting that it may be involved in dimer formation (12, 13). MATERIALS AND METHODSMobility-Shift Assays. Plasmids were prepared and linearized by standard procedures (17). In vitro transcripts were generated by using either SP6 or T7 RNA polymerase, followed by phenol extraction and ethanol precipitation.Translations were carried out with a wheat germ extract (Promega) with [35S]methionine according to the manufacturer's specifications. The [35S]methionine-labeled translation products were analyzed by electrophoresis in a SDS/ 15% polyacrylamide gel. The gel was subsequently fixed in a solution containing 10% (vol/vol) acetic acid and 20% (vol/ vol) metha...

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

“…protein in surface gene expression. The autoregulatory function of the protein in its own promoter has been observed in a number of genes (49,50). In our study, we found that M protein can transactivate the CCAAT box of the major S promoter to regulate surface expression of L, M, and S proteins.…”

Regulation of cellular and viral protein expression by the Epstein-Barr virus transcriptional regulator Zta: Implications for therapy of EBV associated tumors

“…39,40 It is encoded by EBV IE gene BZLF1 and composed of 245 amino acids. The molecular structure of Zta consists of five functional regions: a transcriptional activation domain (aa 1-167) located in the amino terminus of the protein, [41][42][43] a putative regulatory domain (aa 168-177) containing a major phosphorylation site S-173 required for viral replication, 44 a basic DNA binding domain (aa [178][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193][194]) that determines the specificity of DNA recognition and directly interacts with DNA sequence elements, 33,37,45 a coiled-coil domain (aa 195-227) responsible for homodimerization and an accessory activation domain (aa 228-245) in the carboxy terminus.…”

Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter.

“…Zta is in the bZip class of transcription factors (Farrell et al 1989;Chang et al 1990;Flemington and Speck 1990;Lieberman and Berk 1990)• The Zta amino-terminal activation domain is neither acidic nor significantly enriched in any one type of amino acid residue (Giot et al 1991;Lieberman and Berk 1991). With a template containing a TATA box and seven upstream Zta-binding sites, purified Zta stimulated transcription -10-fold in reactions reconstituted with either phosphocellulose D fraction or eTFIID complex (Fig.…”