BIOCHEMICAL/GENETIC ANALYSIS OF HERPESVIRUS ICP4 PROTEIN

伪狂犬病病毒感染细胞将作为模型系统 真核基因转录激活机制的研究。这个 该病毒ICP4蛋白调控早期疱疹病毒的转录 基因并诱导无关腺病毒基因的转录 混合感染。此外，ICP4还激活了不相关的细胞基因 共转染法。因此，ICP4以及腺病毒E1a蛋白， 可能是间接激活转录，而不是通过结合 特定的DNA序列与原核生物的调节蛋白一样。ICP4将是 单抗亲和层析纯化及其生化特性 为理解转录的目的而分析的属性 激活。将使用ICP4的单抗来搜索 ICP4与细胞内转录调控蛋白的相互作用 手机。由于ICP4蛋白是180K的，可能是多功能的， ICP4内的缺失突变体将被制造并重组成病毒以 ICP4生化特性的丧失与ICP4活性的丧失 转录激活功能。最后，我们将继续我们的 温度敏感型ICP4的活性研究 通过确定蛋白质中是否存在不同的基因表达 这种蛋白质可以抑制状态。一种具有非常强的 增强子序列将被导入细胞并感染TSG 不允许的温度来确定强烈增强的基因是否 被禁止了。早期含有整合腺病毒的一系列细胞系 将对基因进行测试以确定染色质中是否存在相同的基因 可被TSG感染所抑制。我们对基因的了解是 激活和提纯蛋白质的能力，这些蛋白质负责 激活给我们提供了一个研究转录的极好的系统 真核细胞中的激活。

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.