HERPESVIRUS GENOMIC CLEAVAGE

疱疹病毒基因组裂解

• 批准号: 6169113
• 负责人: DANIEL E NIXON
• 金额: $ 12.47万
• 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/6169113
• 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.

在完成传染病临床培训后，我的最后一次 7个月来一直在进行基础疱疹病毒研究， 斯图尔特·P·阿德勒博士和迈克尔·麦克沃伊博士的指导， 经验丰富的疱疹病毒研究者。 这一经历使 我有强烈的愿望，追求一个基本的病毒学术生涯 以临床为重点的发病机制研究。 研究设施 这里已经证明足够我完成 拟议项目的初步工作-对 疱疹病毒基因组切割、包装和 循环化。 该项目的具体目标是：1）确定 在切割位点的基本顺式DNA元件，并确定 它们介导的过程：裂解、包装或环化。 在一至两个切割处含有突变的重组gpCMV 将建设场地。 切割、包装或环化 然后确定诱变位点的功能。（二） 纯化、鉴定和表达结合至 CIS元素。 将对两种样品进行凝胶位移试验 感染和未感染的细胞裂解物，以鉴定DNA结合 使用含有确定为 在第1节中是必要的。 DNA结合域会很好 通过寡核苷酸序列的诱变作图。 合成能力 寡核苷酸竞争蛋白质的DNA结合位点， 将测试在组织培养中抑制病毒复制。 结合 蛋白质将在DNA亲和柱上进行纯化，并部分纯化 测序以推断开放阅读框的DNA序列 编码目标蛋白质。 这些序列将用于 构建用于筛选gpCMV cDNA的探针 图书馆 由这些cDNA编码的蛋白质将在大肠杆菌中表达。 重组杆状病毒。 3)测定生化活性 这些蛋白质成分。 活动可能包括特定地点 核酸内切酶活性（切割）DNA末端的连接 （环化）和DNA易位（包装）。 这些 将以两种方式测试功能：（a）生物化学测定， 使用人工质粒底物切割环化，和 杆状病毒表达重组蛋白。 (B)建设 敲除突变的重组病毒和DNA分析 在不存在蛋白质的情况下形成的复制中间体 兴趣 希望这些研究将扩展我们对 这些关键功能的所有疱疹病毒，并协助在 抗病毒剂的发展。 再加上密集的毕业生 层次基础科学课程作业，期刊俱乐部，会议 出席，并与导师和其他人经常互动 调查员，我觉得这个全面的建议将允许我 成为一名独立的调查员。