IN VITRO ASSEMBLY AND INHIBITION OF HIV CAPSIDS

描述：这个项目的目标是定义氨基酸序列

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.