CHARACTERIZATION OF HIV INTEGRASE & ASSOCIATED FACTORS IN MICROBIAL SYSTEMS

HIV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。该反应由整合酶介导 酶（In）通过形成交错的双链断裂 (DSB)在宿主DNA中，病毒DNA末端共价连接到 宿主DNA我们正在开发酵母和E。大肠杆菌基因检测 研究In体内机制的系统，以及 改变反应。 我们已经研究了HIV在野生型和非野生型中过度表达的影响， 各种突变酵母菌株，并已发现没有变化的增长， 形态、活力或对DNA损伤剂的敏感性。增加 我们的检测灵敏度，我们构建了一个有针对性的In融合 蛋白它由酵母GAL4 DNA结合结构域融合到 In蛋白的5'端。其他人的工作表明i）为此目的的融合 的In对于体内测定仍然具有活性，和ii）GAL4 DNA结合 结构域融合蛋白是核定位的。我们正在测试这个 构建不仅在双杂交筛选中，而且在遗传测定中 上面描述我们还准备了一个含有3个重复序列的质粒， 靶序列以检查质粒损失。这些重复也将是 置于两个ura3异等位基因之一的旁边，以确定是否通过 刺激重组。这些试验将用于筛选 人类cDNA、药物和酵母突变体，它们改变了 酵母在大肠我们已经发现，低水平过表达的a 可溶形式的In是致命的。致死之前没有SOS诱导， 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和与In结合的β 1，以确定 In的致死性被RMB1修饰，并检查缺失突变体 以确定哪个区域导致杀戮。

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.