The viral polypeptide designated ICP4 is synthesized during the immediate early phase of productive infection by herpes simples virus (HSV). ICP4 is a positive regulator of early gene transcription and a negative regulator of immediate early gene transcription. Native ICP4 has been partially purified and shown to exist as a dimer which associates directly or indirectly with specific sequences in double-stranded DNA.
The specific aims of this investigation are (i) to obtain a homogeneous preparation of ICP4; (ii) to determine what factors are required to stabilize the association between ICP4 and DNA; (iii) to identify the location and sequence of nucleotides recognized by ICP4 (or associated factors) in the viral genome; (iv) to characterize potential catalytic activities associated with ICP4; (v) to map the location of functional domains in ICP4; (vi) to establish the mechanism for ICP4-mediated repression of transcription; and (vii) to investigate the mechanism for ICP4-mediated induction of transcription. Purification of ICP4 will be achieved by DNA affinity and immunoaffinity chromatography. Both radioimmunoassays and DNA binding assays will be used to quantitate the recovery of ICP4 during purification. The stability of ICP4:DNA complexes will be analyzed by filter binding assays. A library of cloned fragments of HSV DNA will be screened for the presence of ICP4 binding sites by DNA immunoassays. The nucleotide sequences in the binding sites will be identified by DNase footprinting. A computer-assisted homology search will be used to locate related sequences in viral and cellular DNAs. The role that these ICP4 binding sites play in the control of gene transcription will be examined by in vitro transcription assays using templates which contain modified forms of an ICP4 binding site. Fragments of the ICP4 polypeptide will be produced in E. coli by insertion of subgenic portions of the ICP4 gene into bacterial expression vectors. These polypeptide fragments will be reacted with monoclonal antibodies against ICP4 and with radiolabeled DNA in binding reactions designed to map the location of active domains in ICP4. These studies will focus on the long range goal of this investigation, which is to understand the mechanisms which regulate gene expression in mammalian cells. Because regulation of gene expression is a fundamental process that controls the growth, development, and differentiation of human cells, it is important that these mechanisms be understood.
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