The DNA binding domain of the Varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1

Abstract

Varicella-zoster virus gene 62 encodes a protein with predicted Mr of 140,000D (VZV 140k) that shares extensive predicted amino acid sequence homology with the major immediate early (IE) transcriptional regulator protein of herpes simplex virus type 1 (HSV-1) Vmw175. The integrity of highly conserved region 2 is essential for the DNA binding and transcriptional regulatory functions of Vmw175. Similarly, an insertion mutation in region 2 (codons 468-641) of 140k eliminates the transcriptional repression and activation functions of this protein. We have expressed a fragment of 140k which encompasses region 2 as a non-fusion polypeptide in bacteria. This 140k DNA binding domain peptide (codons 417-646) binds to numerous DNA sequences throughout the VZV gene 62 promoter region. It induces multiple regions of protection from DNase I digestion, flanked by sites of DNase I hypersensitivity. Several of the sites recognized can be considered to be divergent forms of the consensus sequence which is recognized by Vmw175. However, by use of a panel of mutagenized probe fragments, we found that the 140k DNA binding domain was less sequence-specific than Vmw175 in its interactions with DNA. Consistent with this, the homologous Vmw175 DNA binding domain, and also intact Vmw175, recognize the gene 62 binding sites much less efficiently than the 140k DNA binding domain. Also in contrast to the situation with Vmw175, the 140k DNA binding domain failed to induce DNA bending when occupying the binding sites in its own promoter. Deletion analysis has mapped the minimal DNA binding domain of the VZV 140k protein, as measured in gel retardation analysis, to lie within residues 472 to 633. The differences in binding characteristics of the DNA binding domains of the homologous VZV 140k and HSV-1 Vmw175 IE proteins may account for the subtle differences in their regulatory activities in transfection assays and during virus growth in tissue culture.

Isolation and initial characterization of the BRCA2 promoter

“…IE62 has only weak DNA binding activity that demonstrates little sequence speci®city (Tyler and Everett, 1993). We demonstrate that the activation with IE62 required an intact E-box and was inhibited by two dierent dominant negative USF-2 constructs.…”

Lessons to be learned from varicella-zoster virus

“…Viral infection is boosted when ORF62p is expressed in cells transfected with ORF62 (Schoonbroodt et al, 1996). ORF62p has distinct functional domains: a powerful activation domain in the N-terminal region (Perera et al, 1993;, a domain at the border between regions 4 and 5 playing possibly a role in promoter activation and repression (Baudoux et al, 1995), a DNA-binding domain in region 2 (Wu and Wilcox, 1991;Tyler and Everett, 1993;Tyler et al, 1994) and a nuclear localization signal in region 3 (Baudoux et al, 1995). ORF62p activates the DNA polymerase and the major DNA binding protein genes of VZV (ORFs 28 and 29), which are juxtaposed in the genome (Meier et al, 1994).…”

An epitope within the DNA-binding domain of the herpes simplex virus immediate early protein Vmw175 is conserved in the varicella-zoster virus gene 62 protein

“…H denotes a host background band which is detected using the Amersham ECL system by most (but not all) of the monoclonal antibodies described in this study. (residues 417 to 646), VT4 (residues 462 to 646) and VT5 (residues 472 to 646) (lanes 2 to 4) were expressed in Escherichia coli (Tyler & Everett, 1993) and detected by Western blotting using monoclonal antibody 10084 at a dilution of 1 : 1000. Lane 1 contains a protein extract from a control E. eoli culture which did not harbour a VZV gene 62 expression plasmid.…”

“…Lane 1 contains a protein extract from a control E. eoli culture which did not harbour a VZV gene 62 expression plasmid. The sizes of the VT2X and VT4 polypeptides are dependent not only on the number of VZV gene 62 residues expressed, but also on the nature of these residues and the number of residues derived from the vector plasmid (Tyler & Everett, 1993).…”

“…These proteins are highly conserved, especially in region 2 (which comprises the majority of the DNA-binding domain) (McGeoch et al, 1986). In the initial experiments, the target for the monoclonal antibodies was protein VT2, which was expressed in bacteria and includes the DNA-binding domain of VZV gene 62 (Tyler & Everett, 1993). A number of the monoclonal antibodies gave a weak reaction against protein VT2 on Western blots, but 10084 was particularly strong, both on blots and in ELISAs (data not shown).…”