DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

Abstract

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to Epstein-Barr virus (EBV) is a lymphotropic human herpesvirus that latently infects B lymphocytes, resulting in a concomitant growth transformation of the infected cell. Infection is closely associated with several human cancers, including nasopharyngeal carcinoma and African Burkitt's lymphoma, and also plays a role in several lymphoproliferative diseases in immunocompromised individuals (for a review, see reference 29). In vitro the transforming potential of EBV is evidenced by its ability to immortalize B lymphocytes to grow indefinitely in culture. Immortalization is achieved through the expression of a relatively small subset of EBV-encoded genes that serve to establish and maintain cellular transformation (for a review, see reference 45).Propagation of EBV from host to host is dependent upon the activation of an estimated 100 or more viral genes, culminating in the production of infectious virions (2, 4, 9, 27). While these genes remain quiescent during latency, a switch in the genetic program leading to the expression of viral replication associated genes can be accomplished in vitro by treatment of latently infected B lymphocytes with various reagents, including phorbol esters, butyrate, ionophore, and anti-immunoglobulin (2,28,37,46,48,51). Activation of the lytic cascade by treatment with anti-immunoglobulin results initially in the expression of two viral genes, BZLF1 and BRLF1, which exhibit similar induction kinetics (maximal mRNA levels are reached between 2 and 4 h postinduction) (14, 46). The protein products of both the BZLF1 (referred to here as Zta, but also called ZEBRA and EB1) and BRLF1 (Rta) genes have been shown to be transcriptional activators (8,10,12,25,26 speck@visar.wustl.edu.Expression of Zta and Rta leads to the activation of early genes and ultimately viral replication. Of all the viral transactivators examined, Zta is unique in that its expression alone can initiate the entire lytic cascade (10,11,40), and regulation of Zta expression appears to be central to regulating entry into the lytic cycle.We have previously identified multiple elements within the BZLF1 promoter (Zp) that play a role in regulating BZLF1 expression (15,16). Several of these elements bind cellular transcription factors that are responsive to phorbol ester stimulation (15). Zp also contains two Zta binding sites (ZIIIA and ZIIIB) that are important for autoactivation (16,33,49).Zta shares several structural similarities with other transcription factors. As described by Farrell et al. (12), Zta contains a basic region with homology to the DNA-binding domains of the AP-1 family of transcription factors (Fig. 1). In addi...

Stimulation of CREB Binding Protein Nucleosomal Histone Acetyltransferase Activity by a Class of Transcriptional Activators

“…Francis and colleagues have identified a single amino acid substitution mutation in Zta (S186A) that abrogates the transcription activation of latent viral chromosomes but has no detectable effect on the transcription activation of transiently transfected reporter plasmids (32). Additionally, a DNA binding defective mutant of Zta could stimulate transcription from a subset of viral promoters, suggesting Zta activates transcription at some promoters by a DNA binding independent mechanism (31). Together, these observations indicate that Zta can regulate transcription by diverse mechanisms, and modulation of CBP HAT activity, as we have described here, may be an important component of Zta-mediated transcription activation.…”

Biophysical Analysis of Natural Variants of the Multimerization Region of Epstein-Barr Virus Lytic-Switch Protein BZLF1

“…BZLF1 has been previously described as a member of the bZip family (2, 5, 7, 13-15, 20, 25, 28). In support of this conjecture, it has been shown that BZLF1 contains adjacent DNA-binding (approximately residues 175 to 195) and multimerization (approximately residues 196 to 245) regions, and the protein interacts with specific DNA sequence elements (2,5,8,13,15,20,28,30) as a multimer (2,8,15). By analogy with other members of the bZip family, multimerization has been assumed to occur through the folding of a coiled-coil interface within this region (i.e., residues 196 to 245).…”

Epstein-Barr Virus Lytic Transactivator Zta Enhances Chemotactic Activity through Induction of Interleukin-8 in Nasopharyngeal Carcinoma Cells

“…7C), indicating binding of Zta to the IL-8 promoter in vivo. We also examined the effects of two DNA-binding-defective Zta mutants, Zdbm1 and Zdbm2, which contain mutations within the DNA-binding domain (28). The two Zta mutants were not recruited to the IL-8 promoter in a ChIP assay (Fig.…”

“…The simian virus 40 promoter-driven, Zta-or Rta-expressing plasmids have been described in our previous study (13). Plasmids expressing Zta with deletion of amino acids 27 to 53 (d27/53) or amino acids 52 to 78 (d52/78), or with mutations within the DNA-binding domain, have also been used previously (27,28). A series of pIL-8-Luc, the reporter plasmids with deletion or site-directed mutation of the IL-8 promoter, were constructed by inserting PCRamplified IL-8 promoter fragments into pGL2-basic vector (Promega) at the 5Ј KpnI site and the 3Ј NheI site.…”