The five human herpesviruses are endemic in the population and are responsible for a variety of clinically significant diseases. These viruses establish life-long infections and four of the five have been associated etiologically with cancer in man. Successful intervention in the viral replicative cycle, latency and transformation will necessitate a better understanding of the functions of individual viral genes and the manner in which gene expression is regulated. The series of studies proposed herein in designed to further our understanding of the role of the immediate-early protein ICP4 in transcriptional regulation ad to identify the functional roles of other immediate-early proteins - ICPs 0, 22, 27 and 47 - in the replicative process. For this purpose, we will compare the properties of ICP4 produced by ts mutants defective in the pre-DNA synthetic (early) function of ICP4 and the newly-idenified post-DNA-synthetic (late) function of ICP4 with that of wild-type ICP4. Mutant and wild-type forms of ICP 4 will be compared with regard to patterns of phosphorylation, poly(ADP-ribosyl)ation, DNA-binding properties, association with other viral and cellular proteins and ability to induce cellular stress proteins - all properties associated with transcriptional regulation. These studies, combined with fine-mapping and sequencing of mutant and wild-type genes for ICP4, should lead to a better definition of the two functions of ICP4. Efforts will then be made to correlate the phenotypic properties of mutant and wild-type forms of ICP4 with ability of early and late mutants to activate transcription from chimeric genes containing selected immediate-early, early, and late viral promoter regulatory sequences. Mutations will also be introduced into these sequences in order to identify important sites recognized by wild-type ICP4. Studies of other immediate-early genes will include 1) genetic and phenotypic characterization of three noncomplementing ts mutants which map in or near the coding sequences for ICP27, 2) introduction of insertion, deletion and ts mutants into the genes for ICPs 0, 22, 27 and 47 by in vitro mutagenesis, and 3) isolation of extragenic suppressor mutations from ts mutants in essential immediate-early genes as a means of identifying other viral proteisn with which immediate-early proteins interact.
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