HERPESVIRUS GENOMIC CLEAVAGE

疱疹病毒基因组裂解

• 批准号: 2671440
• 负责人: DANIEL E NIXON
• 金额: $ 7.95万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2671440
• 关键词: Baculoviridae; DNA binding protein; Herpesviridae; chemical cleavage; circular DNA; endonuclease; gel mobility shift assay; gene mutation; gene targeting; genetic regulatory element; genome; intracellular transport; laboratory rabbit; oligonucleotides; protein purification; protein structure function; recombinant virus; transcription factor; virus assembly; virus genetics; virus infection mechanism; virus protein

After completing my clinical training in infectious diseases, my last 7 months have been spent pursuing basic herpesviral research under the mentorship of Drs. Stuart P. Adler and Michael McVoy, two experienced herpesviral investigators. This experience has instilled me with a strong desire to pursue an academic career in basic viral pathogenesis research with a clinical focus. The research facilities here have proven more than sufficient for me to complete preliminary work on the project proposed- a detailed analysis of the processes of herpesvirus genome cleavage, packaging and circularization. Specific aims of the project are to: 1) Define essential cis DNA elements at cleavage sites and determine the processes they mediate: cleavage, packaging, or circularization. Recombinant gpCMVs containing mutations at one to two cleavage sites will be constructed. Cleavage, packaging, or circularization functions will then be determined for the mutagenized site. 2) Purify, identify, and express the trans acting proteins that bind to the cis elements. Gel shift assays will be performed on both infected and uninfected cell lysates to identify DNA binding proteins using oligonucleotides containing sequences determined to be essential in section 1. DNA binding domains will be fine mapped by mutagenesis of oligo sequences. The ability of synthetic oligonucleotides that compete for DNA binding sites of proteins to inhibit viral replication in tissue culture will be tested. Binding proteins will be purified on DNA affinity columns and partially sequenced to infer the DNA sequences of the open reading frames encoding the protein of interest. These sequences will be used to construct probes that will be used to screen a gpCMV cDNA library. Proteins encoded by these cDNAs will be expressed in recombinant baculoviruses. 3) Determine the biochemical activities of these protein components. Activities may include site specific endonuclease activity (cleavage) ligation of DNA ends (circularization), and DNA translocation (packaging). These functions will be tested in 2 ways: (a) Biochemical assays for cleavage an circularization using artificial plasmid substrates and baculovirus expressed recombinant proteins. (B) construction of recombinant viruses with knockout mutations and analysis of DNA replicative intermediates formed in the absence of the protein of interest. It is hoped that these studies will extend our knowledge of these critical functions of all herpes viruses and assist in the development of antiviral agents. Coupled with intensive graduate level basic science course work, journal club, conference attendance, and frequent interaction with mentors and other investigator, I feel this comprehensive proposal will allow me to become a capable independent investigator.

完成传染病的临床培训后，我的最后一次 我们花了 7 个月的时间进行基础疱疹病毒研究 博士的指导。斯图尔特·P·阿德勒和迈克尔·麦克沃伊，两人 经验丰富的疱疹病毒调查员。 这段经历给我们灌输了 我强烈渴望在基础病毒领域从事学术职业 以临床为重点的发病机制研究。 研究设施 事实证明，这足以让我完成 拟议项目的初步工作——详细分析 疱疹病毒基因组切割、包装和 环化。 该项目的具体目标是： 1) 定义 切割位点的必需顺式 DNA 元件并确定 它们介导的过程：裂解、包装或环化。 重组 gpCMV 在 1 至 2 个裂解处含有突变 将建设站点。 裂解、包装或环化 然后将确定诱变位点的功能。 2） 纯化、鉴定和表达结合的反式作用蛋白 顺式元素。 凝胶位移测定将在两者上进行 感染和未感染的细胞裂解物以鉴定 DNA 结合 使用含有确定序列的寡核苷酸的蛋白质 在第 1 节中是必不可少的。DNA 结合域就可以了 通过寡核苷酸序列的诱变进行定位。 合成能力 竞争蛋白质 DNA 结合位点的寡核苷酸 将在组织培养中测试抑制病毒复制。 装订 蛋白质将在 DNA 亲和柱上纯化，并部分纯化 测序以推断开放阅读框的 DNA 序列 编码感兴趣的蛋白质。 这些序列将用于 构建用于筛选 gpCMV cDNA 的探针 图书馆。 这些 cDNA 编码的蛋白质将在 重组杆状病毒。 3) 生化活性测定 这些蛋白质成分。 活动可能包括特定地点 DNA 末端的核酸内切酶活性（切割）连接 （环化）和 DNA 易位（包装）。 这些 功能将通过两种方式进行测试： (a) 生化测定 使用人工质粒底物切割环化，并 杆状病毒表达重组蛋白。 （二）建设 具有敲除突变的重组病毒和 DNA 分析 在缺乏蛋白质的情况下形成的复制中间体 兴趣。 希望这些研究能够扩展我们的知识 所有疱疹病毒的这些关键功能并有助于 抗病毒药物的开发。 再加上精读毕业生 水平基础科学课程作业、期刊俱乐部、会议 出席，并与导师和其他人频繁互动 调查员，我觉得这个全面的提案将使我能够 成为一名有能力的独立调查员。