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

Abstract

BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV) replication. On the basis of limited sequence homology and mutagenesis experiments, BZLF1 has been described as a member of the bZip family of transcription factors, but this prospect has not been rigorously tested to date. Here, we present biophysical analysis of the multimerization domain of BZLF1, from three natural variants of EBV, and demonstrate for the first time that the region between amino acids 196 and 227 is sufficient to direct folding as a coiled-coil dimer in vitro.BZLF1 (also known as Zebra, Zta, Z, and EB1) is a key component of the machinery that induces the lytic replicative cycle of Epstein-Barr virus (EBV) (3,4,6,22,27). Increased expression of BZLF1 is one of the first events that can be detected following the induction of the lytic cycle in EBVharboring B lymphocytes, and the enforced expression of BZLF1 is sufficient to induce the lytic cycle in cells containing EBV genomes. BZLF1 functions as a transcription factor and activates its own expression and that of a subset of EBV and cellular genes through sequence-specific BZLF1 response elements within their respective promoters. Furthermore, BZLF1 also acts as a replication factor by interacting specifically with the viral lytic origin of replication (23), again through specific BZLF1 response elements (reviewed in references 21 and 24-26). 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). The analysis of deletion mutants of BZLF1 and mutants containing one or more amino acid substitutions at residues within the proposed coiled-coil interface provided some support for this model (2, 5, 7, 13-15, 20, 25, 28), although there has been disagreement about the influence of some residues (Y200 and L225, for example) on the ability to multimerize (7,12). Therefore, while the results of numerous studies have been consistent with the existence of a coiled-coil interface on BZLF1, the studies have fallen short of rigorously testing the model.To date, three naturally occurring sequence variants within the multimerization domain of BZLF1 have been described. Previous analyses have focused on the BZLF1 sequence deduced from the B95-8 isolate of EBV (1); using this as a reference sequence, the two variants are A205S (19) and A206S (10). In this study, we sought to determine whether further variants of the multimerization region occur in natural isolates of EBV before exploring biophysically whether BZLF1 is indeed able to form multimers through a coiled-coil inte...

bZIP proteins of human gammaherpesviruses

“…Zta interacts with a series of related DNA-binding sites termed Zta-response elements (ZREs). Recently, it has been demonstrated that the dimerization domain of Zta folds as a coiled-coil, although the strength of the interaction is much weaker than that of other bZIP proteins (Hicks et al, 2001). The mechanism by which Zta achieves stability as a dimer awaits further investigation.…”

Investigation of the Multimerization Region of the Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Protein K-bZIP: the Proposed Leucine Zipper Region Encodes a Multimerization Domain with an Unusual Structure

“…We questioned whether this occurs by using two biophysical approaches. A synthetic peptide was generated that was equivalent to the region of Zta that we had previously shown to fold as a coiled coil in vitro (16). The propensity of the K-bZIP peptide to fold as an ␣-helix was assessed first using circular dichroism (CD) spectroscopy.…”

Role of the Epstein-Barr Virus ZEBRA Protein and HPV in the Carcinogenesis of Nasopharyngeal Carcinoma

“…By analogy with other members of the bZIP family, the multimerization interface of ZEBRA has been predicted to fold through a coiled-coil structure (Sinclair & Farrell 1992). Biophysical evidence that this prediction holds true was recently provided (Hicks et al 2001). …”