HERPESVIRUS GENOMIC CLEAVAGE

在完成了我的传染病临床培训后，我的最后一次 已经花了7个月的时间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调查员。这段经历给我们灌输了 我有强烈的愿望去追求Basic Virus的学术生涯 病机研究以临床为重点。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 疱疹病毒基因组的切割、包装和加工过程 循环化。该项目的具体目标是：1)确定 切割位点上的基本顺式DNA元件并确定 它们所调节的过程：卵裂、包装或环化。 含有一到两个裂解突变的重组gpCMV 将建造场地。卵裂、包装或环化 然后将确定突变位点的功能。2) 纯化、鉴定和表达与其结合的反式作用蛋白 顺式元素。将对这两种情况进行凝胶移位分析 感染和未感染细胞裂解物鉴定DNA结合 使用含有确定的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 通过对寡核苷酸序列的突变来定位。人的综合能力 竞争蛋白质的DNA结合部位的寡核苷酸 在组织培养中抑制病毒复制将进行测试。装订 蛋白质将在DNA亲和层析柱上纯化，并部分 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 构建可用于筛选gpCMV基因的探针 图书馆。由这些cDNA编码的蛋白质将在 重组杆状病毒。3)生化活性的测定 这些蛋白质成分中。活动可能包括现场特定活动 DNA末端的核酸内切酶活性(切割)连接 (环化)和DNA移位(包装)。这些 功能测试将通过两种方式进行：(A)生化分析 使用人工质粒底物切割环状结构 杆状病毒表达重组蛋白。(B)建造 基因敲除突变重组病毒及其DNA分析 在没有蛋白质的情况下形成的复制中间体 利息。希望这些研究将扩大我们对 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。再加上密集的毕业生 层次基础科学课程工作，期刊俱乐部，会议 出席，并与导师和其他人频繁互动 调查员，我觉得这份全面的提案能让我 成为一名有能力的独立调查员。

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.