IN VITRO ASSEMBLY AND INHIBITION OF HIV CAPSIDS

描述：该项目的目标是定义氨基酸序列 在可与HIV Gag前体结合并与其结合的随机多肽库中 建立一种可用于测试这些潜力的体外系统 艾滋病毒组装的竞争性抑制物。这些分子将提供 洞察衣壳组装和衣壳的重要结构域 可以形成未来设计的先导化合物的抑制剂 组装过程。在本申请中，申请人提出：1. 膜非依赖体外翻译/组装系统的研制 针对HIV GAG，基于已经为梅森-辉瑞猴子建立的系统 病毒(M-PMV)Gag.嵌合HIV Gag前体将被构建为 含有M-PMV Gag序列，在没有M-PMV的情况下促进体外组装 膜。 2.GAG前体结合多肽抑制剂的发现 利用酵母双杂交系统和随机多肽表达文库。 3.能与GAG特异结合的多肽的鉴定 前体、CA-NC和CA使用随机多肽文库，这就是 珠一肽“(选择肽)组合文库技术。潜力 通过两个筛查确定的抑制剂将被测试对病毒的效果 培养中的复制。 4.GAG结构和组装结构域的分子遗传学分析 多肽相互作用。结构构件的可能位置， 由结合多肽识别，参与组装将通过以下方式探测 体外组装试验和体外组装试验中的定向突变及分析 在表达研究和病毒传播试验的背景下。

DESCRIPTION: The goals of this project are to define amino acid sequences in random peptide libraries that can bind to the HIV Gag precursor and to establish an in vitro system which can be used to test these potential competitive inhibitors of HIV assembly. These molecules will provide insight into important structural domains involved in capsid assembly and could form the lead compounds for the future design of inhibitors of the assembly process. In this application the applicant proposes: 1. Development of a membrane independent in vitro translation/assembly system for HIV Gag, based upon a system already established for Mason-Pfizer monkey virus (M-PMV) Gag. Chimeric HIV Gag precursors will be constructed to contain M-PMV Gag sequences that promote assembly in vitro in the absence of membranes. 2. Discovery of peptide-based inhibitors of Gag precursors association using a yeast two hybrid system and random peptide expression libraries. 3. Identification of peptides that can specifically associate with the Gag precursor, CA-NC and CA using random peptide libraries, that is the "one bead one peptide" (Selectide) combinatorial library technology. Potential inhibitors identified by both screens will be tested for effect upon virus replication in culture. 4. Molecular genetic analysis of Gag structure and assembly domains by peptide interactions. The probable locations of structural elements, identified by binding peptides, involved in assembly will be probed by directed mutagenesis and analysis in both the in vitro assembly assay and in vivo in the context of expression studies and virus spread assays.