BIOCHEMICAL/GENETIC ANALYSIS OF HERPESVIRUS ICP4 PROTEIN

伪狂犬病病毒感染的细胞将被用作模型系统， 研究真核生物基因转录的激活机制。 的 该病毒的ICP 4蛋白调节早期疱疹病毒的转录 基因，并诱导无关腺病毒基因的转录， 合并感染 此外，ICP 4还激活了细胞中不相关的细胞基因。 共转染测定。 因此，ICP 4以及腺病毒E1 A蛋白， 可能是间接激活转录，而不是通过结合 特异性DNA序列，如原核调节蛋白所做的。 ICP 4将 通过单克隆抗体亲和层析纯化， 为了理解转录的目的而分析的性质 activation. ICP 4的单克隆抗体将用于检索 ICP 4和转录调节蛋白之间的相互作用 cell. 由于ICP 4蛋白质是180 K并且可能是多功能的， 将制备ICP 4内的缺失突变体并重组到病毒中， 将ICP 4的生物化学性质的损失与ICP 4的生物化学性质的损失相关联。 转录激活功能 最后，我们将继续 温度敏感型ICP-4活性的研究 蛋白质通过确定基因的表达是否存在于不同的 这种蛋白质可以抑制这种状态。 一种基因， 将增强子序列转染到细胞中并用tsG感染， 非允许的温度，以确定是否强烈增强的基因， 压抑 一系列含整合腺病毒的细胞系早期 基因将被测试，以确定相同的基因是否存在于染色质中， 可被tsG感染抑制。 我们对基因的了解 激活和纯化蛋白质的能力， 激活给了我们一个很好的系统来研究转录 在真核细胞中激活。

Pseudorabies virus infected cells will be used as a model system for studying the mechanism of activation of eukaryotic gene transcription. The ICP4 protein of this virus regulates the transcription of early herpesvirus genes and induces the transcription of unrelated adenovirus genes in coinfections. In addition, ICP4 activates unrelated cell genes in cotransfection assays. Thus ICP4, as well as the adenovirus E1A protein, may be activating transcription indirectly, rather than by binding to specific DNA sequences as prokaryotic regulatory proteins do. ICP4 will be purified by monoclonal antibody affinity chromatography and its biochemical properties analyzed for the purpose of understanding transcriptional activation. Monoclonal antibodies to ICP4 will be used to search for interactions between ICP4 and transcriptional regulatory proteins of the cell. Since the ICP4 protein is 180K and possibly multifunctional, deletion mutants within ICP4 will be made and recombined into virus to correlate the loss of biochemical properties of ICP4 with the loss of the transcriptional activation function. Finally, we will continue our investigations into the activities of the temperature sensitive ICP4 protein by determining whether the expression of genes present in different states can be inhibited by this protein. A gene with a very strong enhancer sequence will be transfected into cells and infected with tsG at the nonpermissive temperature to determine if strongly enhanced genes are inhibited. A series of cell lines containing integrated adenovirus early genes will be tested to determine if the same genes present in chromatin can be repressed by tsG infection. Our knowledge of the genes which are activated and the ability to purify the protein responsible for their activation gives us an excellent system for studying transcriptional activation in eukaryotic cells.