Characterisation of the DNA Binding Domain of the Transcriptional Regulator Encoded by VZV Gene 62

Tyler, Jessica Kate (1994) Characterisation of the DNA Binding Domain of the Transcriptional Regulator Encoded by VZV Gene 62. PhD thesis, University of Glasgow.

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The understanding of gene regulation during varicella-zoster virus (VZV) infection is still rudimentary, although insight can be gained from the more extensively studied system of the genetically related herpes simplex virus type 1 (HSV-1). Upon infection, HSV-1 expresses three main classes of genes in a coordinately regulated temporal cascade; the immediate-early (IE), early (E) and late (L) genes. The product of the HSV-1 IE3 gene, Vmwl75, is considered to be the major regulatory protein of HSV-1 because functional Vmwl75 is an absolute requirement for transactivation of E and L gene expression and for the down-regulation of its own IE3 promoter. The polypeptide encoded by VZV gene 62 (VZV 140k) is the sequence homologue of HSV-1 Vmwl75. Transient transfection experiments have demonstrated that VZV 140k is a potent and promiscuous transactivator of gene expression, and that it can also negatively autoregulate its own gene 62 promoter. In addition, VZV 140k can complement HSV-1 viruses lacking functional Vmwl75. Therefore, by analogy to its HSV-1 counterpart, VZV 140k is likely to play a critical role in the regulation of VZV gene expression. The research described in this Thesis aimed to investigate the properties and activities of the DNA binding domain of the VZV 140k protein, in order to gain a better understanding of the mechanisms of 140k-mediated gene regulation during infection. The primary sequences of VZV 140k and HSV-1 Vmwl75 have been divided into five co-linear regions on the basis of the extent of their predicted amino acid identity. The region 2 sequences are particularly highly conserved between the two proteins and the regulatory functions of Vmwl75 require the integrity of region 2. Furthermore, sequences spanning HSV-1 Vmwl75 region 2 constitute a physically separable, stable DNA binding domain. Many of the experiments in this study stemmed from the initial demonstration that the corresponding region of the VZV 140k protein also yields a sequence-specific DNA binding domain when expressed as a non-fusion polypeptide in bacteria. The bacterially expressed VZV 140k DNA binding domain specifically recognised multiple DNA sequences in the vicinity of its target promoters; the 140k binding sites identified within its own VZV gene 62 promoter may have a role in the negative autoregulatory function of the 140k protein. Several of the 140k recognition sites showed sequence similarity to the Vmwl75 consensus binding sequence, although gel retardation assays with a systematically mutagenised binding site found the VZV 140k DNA binding domain peptide to be less sequence-specific than the corresponding domain of HSV-1 Vmwl75. These and further differences that have been identified between the DNA binding activities of the VZV 140k and HSV-1 Vmwl75 DNA binding domains may explain the previously reported variations between the regulatory functions of 140k and Vmwl75. The minimal region of the VZV 140k protein required for sequence-specific DNA binding has been defined by a deletion analysis to within 162 residues, essentially comprising the highly conserved region 2. However, additional sequences from the C-terminal end of region 1 were necessary for the full DNA binding interaction, as determined by DNase I footprinting analysis. As such, the DNA binding domain of VZV 140k is larger than those reported for other transcriptional regulators (which are often less than 80 amino acids) and therefore it may constitute a novel type of DNA binding structure. A short sequence that exhibits intriguing homology to the conserved DNA recognition helix of the homeodomain DNA binding motif is found at the centre of the VZV 140k DNA binding domain. The alterations to the DNA binding interaction that resulted from the substitution of single amino acids within this region indicate its probable involvement in the DNA recognition by VZV 140k. Of particular note, mutation of a single lysine residue drastically reduced the DNA binding activity and destroyed the transactivation function of VZV 140k; this result emphasises the importance of DNA binding for VZV 140k-mediated gene regulation. The highly purified VZV 140k DNA binding domain was a stable dimer in solution, as demonstrated by gel filtration, glycerol gradient centrifugation and cross-linking techniques. The fact that the VZV 140k DNA binding domain binds to DNA as a dimer was indicated by gel retardation assays that showed novel protein-DNA complexes of intermediate mobility following in vitro co-translation of pairs of VZV 140k peptides of differing sizes. In addition, the VZV 140k and HSV-1 Vmwl75 DNA binding domain peptides readily heterodimerised on co-translation, which indicated that these two related domains have similar modes of dimerisation. In vitro co-translation of a wide range of truncation and insertion mutation versions of the VZV 140k and HSV-1 Vmwl75 DNA binding domains, followed by analysis of their dimerisation by co-immunoprecipitation and their DNA binding ability by gel retardation, has indicated that the structural requirements for dimerisation are lower than for the DNA binding interaction. Finally, construction of a hybrid Vmwl75 protein with the VZV 140k DNA binding domain replacing that of HSV-1 Vmwl75 has allowed the relatedness of the two domains to be assessed directly. The hybrid protein was functional in tissue culture transient transfection assays and furthermore, supported viral growth when expressed from a recombinant HSV-1 genome. The details of the regulatory activities of the hybrid protein in the transient assays implied that the characteristic DNA binding activities of the VZV 140k DNA binding domain play an important part in determining its specific regulatory functions.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John H Subak-Sharpe
Keywords: Virology, Genetics
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75607
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:18
Last Modified: 19 Nov 2019 19:18

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