IN VITRO ASSEMBLY AND INHIBITION OF HIV CAPSIDS

描述：该项目的目标是定义氨基酸序列 在随机肽库中，可以结合HIV Gag前体， 建立一个体外系统，可以用来测试这些潜力 HIV装配的竞争性抑制剂。 这些分子将提供 深入了解参与衣壳组装的重要结构域， 可以形成未来设计抑制剂的先导化合物， 组装过程。 在本申请中，申请人提出：1. 非膜依赖性体外翻译/组装系统的建立 对于HIV Gag，基于已为Mason-Pfizer猴建立的系统 病毒（M-PMV）Gag.嵌合的HIV Gag前体将被构建， 含有M-PMV Gag序列，该序列在缺乏 膜。 2. 发现基于肽的Gag前体缔合抑制剂 使用酵母双杂交系统和随机肽表达文库。 3. 可与Gag特异性结合的肽的鉴定 前体，CA-NC和CA使用随机肽库，这是“一个 珠单肽”（Selectide）组合文库技术。 潜在 将测试两种筛选鉴定出的抑制剂对病毒的作用。 文化中的复制。 4. Gag结构和组装结构域的分子遗传学分析 肽相互作用 结构元素的可能位置， 通过结合肽鉴定，参与组装的肽将通过 在体外组装测定和体外重组测定中的定向诱变和分析 在表达研究和病毒扩散测定的背景下体内。

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.