Human Immunodeficiency Virus Proteinase

描述(申请人提供)：联合应用抗逆转录病毒疗法 RT和PR抑制剂的出现对HIV-1的管理产生了深远的影响 感染。然而，在药物压力下，病毒能够进化为 对现有的药物治疗方案产生抗药性的人。因此，抗药性具有 成为艾滋病治疗面临的最大挑战。我们一直在研究一个 儿童人群中使用蛋白水解酶抑制剂进行临床试验的情况 与RT抑制剂联合应用。我们发现，人类的进化 蛋白水解酶序列伴随着裂解序列的进化 GAG/POL内的站点，从而影响处理效率。此外， 蛋白酶的进化伴随着裂解的变化。 专一性。我们假设Gag/Pol裂解位点和PR形成一个 序列进化可能相互依赖的功能单位。在我们的 续约期内，我们将利用这些发现来获得更多 了解GAG/POL处理的机制。为此，我们将 追求以下具体目标：具体目标我将分析自然恶作剧/POL 在细菌表达中产生的处理表型的等位基因 当前支持期限。我们还将通过准备 嵌合Gag/Poll重组病毒的构建及复制分析 天然或嵌合的GAG/POL区域。《特定目标2》将研究 重组病毒在存在抗蛋白酶药物的情况下培养。在……里面 此外，新的蛋白酶等位基因将被亚克隆和表达，以供分析 使用底物组合文库研究底物专一性， 并通过对抑制剂结合情况的分析，得出结论。具体目标3将研究 GAG加工位点序列的变化对GAG加工效率的影响 正在处理。我们将制备代表GAG/POL产品的多肽 加工并测试它们抑制酶的能力。我们还将进行 Gag/Pol蛋白与HIV灭活型融合蛋白的结构分析 为了确定组织结构在公关过程中的作用 事件。

DESCRIPTION (Provided by applicant): Antiretroviral therapy with a combination of RT and PR inhibitors has had a profound effect upon the management of HIV-1 infection. However, under drug pressure, the virus is able to evolve into forms that are resistant to the available drug regimens. Thus, drug resistance has become the most significant challenge to AIDS therapy. We have been studying a pediatric population undergoing a clinical trial with protease inhibitors in combination with RT inhibitors. We have discovered that evolution of the protease sequence is accompanied by evolution of the sequence of the cleavage sites within Gag/Pol, thus affecting the efficiency of processing. Furthermore, the evolution of protease is accompanied by alterations in cleavage specificity. We hypothesize that the Gag/Pol cleavage sites and PR form a functional unit in which sequence evolution may be co- dependent. In our renewal period, we will exploit these discoveries to gain additional understanding of the mechanisms of Gag/Pol processing. To this end, we will pursue the following specific aims: Specific Aim I will analyze natural Gag/Pol alleles for processing phenotype in a bacterial expression developed in the current period of support. We will also map the determinants by preparation of chimeric gag/pol constructs and analyze replication of recombinant virus with natural or chimeric gag/pol regions. Specific Aim 2 will study the growth of recombinant virus in the presence of anti-protease drugs in culture. In addition, new protease alleles will be subcloned and expressed for analysis by studies of substrate specificity using a combinatorial library of substrates, and by analysis of the binding of inhibitors. Specific Aim 3 will study the effects of changes in the sequence of Gag processing sites on the efficiency of processing. We will prepare peptides representing products of Gag/Pol processing and test their ability to inhibit the enzyme. We will also conduct structural analyses of fusions of Gag/Pol proteins with an inactive form of HIV PR in order to determine the role of structural organization in the processing events.