Tyler J K, Everett R D
MRC Virology Unit, Glasgow, UK.
Nucleic Acids Res. 1993 Feb 11;21(3):513-22. doi: 10.1093/nar/21.3.513.
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.
水痘带状疱疹病毒基因62编码一种预测分子量为140,000D的蛋白质（VZV 140k），它与单纯疱疹病毒1型（HSV-1）的主要即刻早期（IE）转录调节蛋白Vmw175具有广泛的预测氨基酸序列同源性。高度保守区域2的完整性对于Vmw175的DNA结合和转录调节功能至关重要。同样，140k蛋白区域2（密码子468 - 641）中的插入突变消除了该蛋白的转录抑制和激活功能。我们在细菌中表达了包含区域2的140k片段作为非融合多肽。这个140k DNA结合结构域肽（密码子417 - 646）与水痘带状疱疹病毒基因62启动子区域的众多DNA序列结合。它诱导了多个免受DNase I消化的保护区域，两侧是DNase I高敏位点。几个被识别的位点可被认为是Vmw175识别的共有序列的不同形式。然而，通过使用一组诱变的探针片段，我们发现140k DNA结合结构域在与DNA相互作用时比Vmw175的序列特异性更低。与此一致的是，同源的Vmw175 DNA结合结构域以及完整的Vmw175识别基因62结合位点的效率远低于140k DNA结合结构域。同样与Vmw175的情况相反，140k DNA结合结构域在占据其自身启动子中的结合位点时未能诱导DNA弯曲。缺失分析已将凝胶阻滞分析中测量的VZV 140k蛋白的最小DNA结合结构域定位在残基472至633内。同源的VZV 140k和HSV-1 Vmw175 IE蛋白的DNA结合结构域结合特性的差异可能解释了它们在转染试验和组织培养中病毒生长期间调节活性的细微差异。
