The goal of the studies described in this proposal is to elucidate the mechanism by which gag (core) proteins of the human immunodeficiency virus (HIV- 1) are transported to the plasma membrane of infected cells and assemble into virus particles. Our proposal is of direct medical significance because it focuses of a potentially inhibitable step in the life cycle of a lethal human pathogen. In particular, examination of the HIV gag transport/ assembly process may reveal virus specific steps in transport, membrane binding, and budding which may be blocked without significantly perturbing essential cellular processes. Additionally, our proposal addresses basic unresolved questions concerning enveloped animal virus assembly, targeting of myristylated proteins, peripheral membrane protein interactions, and virus budding. The methods we developed for the study of murine retrovirus assembly will be used in our analysis of HIV.
Our specific aims are as follows: 1. Generation of HIV-1 gag mutants: A limited number of HIV gag protein deletion, insertion, and point mutants will be constructed for analysis in a modified wild type (wt) HIV backbone. We will focus on matrix (MA), capsid (CA), and p6 domains with the intention of identifying mutants involved in the assembly process.2. Screening of mutants for replication defective phenotypes: Gag mutants in a HIV construct carrying a selectable marker will be characterized following transfection into primate cells. Phenotypically wt constructs will be identified by scoring transduction of the selectable marker to human target cells. Assembly mutants will be identified by screening for intracellular p55 gag, and examination of released particles by immunoblotting, reverse transcriptase (RT) assay, electron microscopy (EM), and viral RNA analysis. 3.Characterization of assembly mutants. Gag mutants which are defective in virus assembly will be characterized in detail. Subcellular localization studies of HIV wt and mutant gag proteins will employ fractionation and immunofluorescence protocols. Intracellular particle formation will be monitored by EM, and fractionation in the presence of nonionic detergent. With a limited number of mutants, isolation and mapping of second site revertants will be attempted.
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