Hyperacetylation of Biologically Active SV40 Chromatin

描述（由申请人提供）：使用猿猴病毒40（SV 40）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进行的染色质重塑与进入的病毒基因组活化之间的关系，以进行早期转录。本研究的具体目的是：1.确定感染早期SV 40染色体中含有修饰组蛋白的核小体的基因组结构; 2.确定含有修饰的组蛋白的SV 40染色体是否充当调节因子的底物;和3.确定单个调控序列或调控序列的组合是否可以用特定的“组蛋白密码”标记染色质。“来自感染野生型776株病毒或病毒构建体的细胞的SV 40染色质将在感染后30分钟或3小时分离，所述病毒构建体含有引入我们开发的染色质报告病毒的潜在调控序列的各种组合。SV 40染色质将作为完整染色体或在超声处理或微球菌核酸酶消化后进行分析，以通过染色质免疫沉淀（ChIP）试验和竞争性、多重或实时PCR的组合生成适当大小的染色质片段。通过分析由微球菌核酸酶消化产生的单核小体，将确定含有修饰的组蛋白的染色质在SV 40基因组上的具体位置。将使用识别超乙酰化组蛋白、甲基化组蛋白和病毒结构蛋白的抗体，我们在初步研究中已经证明所有这些蛋白在这段时间内与SV 40染色体相关，以及针对已知与早期转录相关的其他形式的组蛋白修饰和转录因子的抗体。许多DMA病毒，虽然不是SV 40，是重要的人类病原体。了解DNA病毒感染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.