CONTROL OF VIRAL RNA SYNTHESIS IN HERPES VIRUS INFECTION

HSV启动子和细胞基本转录机制之间相互作用的不同稳定性以及病毒介导的转录机制的区分化，在很大程度上解释了不同动力学类病毒基因在生产复制周期的不同阶段选择性表达和抑制的原因。我们将通过完成以下工作来扩展和验证该模型：1.分析一类含有下游激活序列(DAS)的严格晚期启动子(以UL38启动子为例)与多功能细胞酶DNA依赖的磷酸激酶(DNA-PK)的DNA结合亚基(Ku)之间的生化相互作用。这项研究的一个主要特点是使用培养细胞，其中DNA-PK的成分已被功能性删除，并纯化了TFIID。2.选择模型启动子来研究HSV用来直接稳定晚期启动子与TFIID复合体之间相互作用的其他方式。将为这项研究开发HSV DNA微阵列。3.研究HSV转录本的动力学类特异性启动子结构如何影响预启动复合体的结合强度，从而影响最大表达时间。我们将使用纯化的TFIID进行生化研究，以及使用原位杂交方法分析单个细胞中的差异基因表达。4.利用细胞培养和小鼠致病模型研究所选病毒基因最大表达时间和水平的确切作用。这项工作始于病毒表达主要衣壳蛋白(VP5)的动态变化。我们还将研究VP19衣壳蛋白、病毒粒子反式激活蛋白和即刻早期ICP27启动子表达的动力学变化。

The differential stability of interactions between kinetic classes of HSV promoters and the basal transcriptional machinery of the cell combined with virus-mediated compartmentalization of transcription machinery does much to explain the selective expression and repression of various kinetic classes of viral genes during the different phases of the productive replication cycle. We will expand and validate this model by accomplishing the following: 1. Analyze the biochemical interaction between a class of strict late promoters that contain a downstream activating sequence (DAS), as exemplified by the UL38 promoter, and the DNA binding subunits (Ku) of the multifunctional cellular enzyme DNA-dependant phosphokinase (DNA-PK). A major feature of this investigation will be the use of cultured cells in which components of DNA-PK have been functionally deleted, and purified TFIID. 2. Choose model promoters to investigate other modes that HSV utilizes to directly stabilize the interaction between late promoters and the TFIID complex. HSV DNA micro-arrays will be developed for this study. 3. Investigate how the kinetic class-specific promoter structure of HSV transcripts influences the strength of binding of the pre-initiation complex to influence time of maximal expression. We will use purified TFIID for biochemical studies, as well as in situ hybridization methods for analysis of differential gene expression in individual cells. 4. Use cell culture and mouse pathogenesis models to study of the precise role of time and level of maximal expression of selected required viral genes. This work has been started with viruses expressing kinetic alterations in the major capsid protein (VP5). We will also study kinetic modifications of expression of the VP19 capsid protein, the virion trans- activating protein, and the immediate-early ICP 27 promoter.