Hyperacetylation of Biologically Active SV40 Chromatin

描述（由申请人提供）：利用猿猴病毒40 （SV40）作为DNA病毒感染的模型，拟议的研究将有助于我们理解通过组蛋白修饰的染色质重塑与进入病毒基因组的早期转录激活之间的关系。本研究的具体目的是：1。确定感染早期SV40染色体中含有修饰组蛋白的核小体的基因组组织；2. 确定含有修饰组蛋白的SV40染色体是否作为调控因子的底物；和3。确定单个调节序列或调节序列的组合是否可以用特定的“组蛋白代码”标记染色质。从被野生型毒株776病毒感染的细胞中提取的SV40染色质或将含有各种潜在调控序列组合的病毒构建物引入我们开发的染色质报告病毒中，将在感染后30分钟或3小时分离。SV40染色质将作为完整的染色体进行分析，或在超声或微球菌核酸酶切后进行分析，通过染色质免疫沉淀（ChIP）测定和竞争、多重或实时PCR相结合产生合适大小的染色质片段。通过分析由微球菌核酸酶酶切产生的单核小体，将确定含有修饰组蛋白的染色质在SV40基因组上的具体位置。抗体将用于识别高乙酰化组蛋白、甲基化组蛋白和病毒结构蛋白，所有这些我们在初步研究中已经证明与SV40染色体相关，抗体将用于识别其他形式的组蛋白修饰和已知与早期转录相关的转录因子。许多DMA病毒，虽然不是SV40，是重要的人类病原体。了解DNA病毒感染的最初几个小时内发生的事件可能最终导致旨在限制这些病毒感染水平传播的治疗干预措施。

DESCRIPTION (provided by applicant): Using simian virus 40 (SV40) as a model for infections by DNA viruses, the proposed research will contribute to our understanding of the relationship between chromatin remodeling through histone modification and the activation of an incoming viral genome for early transcription. The specific aims of the research are: 1. To determine the genomic organization of nucleosomes containing modified histones in SV40 chromosomes early in infection; 2. To determine if SV40 chromosomes containing modified histones serve as substrates for regulatory factors; and 3. To determine whether individual regulatory sequences or combinations of regulatory sequences can label chromatin with a specific "histone code." SV40 chromatin from cells infected with wild-type strain 776 virus or viral constructs containing various combinations of potential regulatory sequences introduced into a chromatin reporter virus which we have developed will be isolated at 30 minutes or 3 hrs post-infection. The SV40 chromatin will be analyzed either as intact chromosomes or following sonication or micrococcal nuclease digestion to generate suitably sized fragments of chromatin by a combination of chromatin immunoprecipitation (ChIP) assays and competitive, multiplex, or real-time PCR. By analyzing mononucleosomes generated by micrococcal nuclease digestion, the specific location of chromatin containing modified histones on the SV40 genome will be determined. Antibodies will be used which recognize hyperacetylated histones, methylated histones, and viral structural proteins, all of which we have shown to be associated with SV40 chromosomes during this time period in preliminary studies, and antibodies to other forms of histone modifications and transcription factors known to be associated with early transcription. Many DMA viruses, although not SV40, are significant human pathogens. Understanding the events occurring during the first hours of infection by DNA viruses may ultimately lead to therapeutic interventions designed to limit the horizontal spread of these viral infections.