CHARACTERIZATION OF HIV INTEGRASE & ASSOCIATED FACTORS IN MICROBIAL SYSTEMS

HIV 整合酶的特征

• 批准号: 2574431
• 负责人: M A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2574431
• 关键词: DNA binding protein; DNA damage; DNA repair; Escherichia coli; alleles; chimeric proteins; drug screening /evaluation; enzyme mechanism; fungal genetics; gene expression; genetic strain; human genetic material tag; human immunodeficiency virus; integrase; mutant; plasmids; virus DNA; yeasts

An obligate step in the life cycle of HIV is the integration of the viral DNA into the host chromosome. This reaction is mediated by the integrase enzyme (In) through the formation of a staggered double strand break (DSB) in the host DNA and covalent linkage of the viral DNA ends to the host DNA. We are developing yeast and E. coli based genetic detection systems to investigate in vivo mechanisms of In, as well as means of altering the reaction. We have examined the effects of overexpression of HIV in wild-type and various mutant yeast strains and have found no change in growth, morphology, viability, or sensitivity to DNA damaging agents. To increase the sensitivity of our assays we constructed a targeted In fusion protein. This consists of the yeast GAL4 DNA binding domain fused to the 5' end of the In protein. Work by others has shown i)fusions to this end of In are still active for in vivo assays, and ii) that GAL4 DNA binding domain fusion proteins are nuclear localized. We are testing this construct not only in two-hybrid screens, but also in the genetic assays described above. We are also preparing a plasmid with 3 repeats of the target sequence to examine plasmid loss. These repeats will also be placed next to one of two ura3 heteroalleles to determine if cutting by In stimulates recombination. These assays will be used to screen for human cDNAs, drugs, and yeast mutants that alter the In reaction in yeast. In E. coli we have found that low level over-expression of a soluble form of In is lethal. Lethality is not preceded by SOS induction, and overexpression of In mutants is also lethal. We are currently co-expressing In and Ini1 (which binds to In) in cells to determine if the lethality of In is modified by Ini1 and examining deletion mutants of In to determine which region causes killing.

艾滋病毒生命周期中的一个必要步骤是整合病毒 DNA进入宿主染色体。该反应是由整合酶介导的 酶(In)通过形成交错的双链断裂 宿主DNA中的DSB和病毒DNA的共价键末端 宿主DNA。我们正在开发基于酵母菌和大肠杆菌的基因检测 用于研究In的体内机制的系统，以及 改变反应。 我们研究了野生型和野生型HIV过度表达的影响 各种突变的酵母菌株，没有发现生长的变化， DNA损伤剂的形态、活性或敏感性。增加 我们构建的核聚变靶点检测的敏感性 蛋白。这是由酵母GAL4 DNA结合域与 In蛋白的5‘末端。其他人的工作表明，i)融合了这一目标 对于体内检测仍然有效，以及ii)GAL4 DNA结合 结构域融合蛋白定位于核。我们正在测试这一点 不仅在双杂交筛选中构建，而且在遗传分析中也构建 如上所述。我们还在准备一种含有3个重复的 检测质粒丢失的靶序列。这些重复也将是 放置在两个ura3异等位基因之一的旁边，以确定是否通过 In刺激重组。这些化验结果将被用于筛查 改变In反应的人类DNA、药物和酵母突变体 酵母。在大肠杆菌中，我们发现了一种低水平的过表达 可溶形式的In是致命的。致死之前并不是SOS诱导， 而In突变体的过度表达也是致命的。我们目前正在 在细胞中共表达In和Ini1(它与In结合)以确定是否 In的致死性被Ini1修饰并检测缺失突变体 以确定哪个区域导致了杀戮。