Although the structural biology of retroviruses, and in particular of HIV-1, has been pursued for years, many fundamental questions remain unanswered. In particular an in depth understanding is lacking of how the Gag protein is targeted to, and interacts with, the inner leaflet of the plasma membrane (PM). Also, the nature of the Gag lattice that forms at assembly sites underneath the plasma membrane is poorly characterized at a molecular level. This renewal application describes experiments that address these two topics. The application, which is intended as a transition to termination grant, seeks modest funding for a three year period. Many of the proposed experiments are either already in progress or are collaborations in which we supply proteins to other labs. For the latter, we envision our role as catalyzing significant science that may then be continued independently by other labs.
Specific Aim 1 of the proposal is to understand how HIV-1 and Rous sarcoma virus (RSV) Gag proteins interact with membranes. We will extend to inner leaflet lipids our observations on the stimulating effect of cholesterol and certain acyl chains on liposome binding. We will dissect the roles of the MA and NC domains in liposome interaction, and will quantify the effects of multimerization. Finally, we will test one hypothesis that might explain th stimulatory effect of cholesterol on membrane binding of Gag, and in a collaboration will determine if MA and Gag proteins prefer positively or negatively curved membranes.
Specific Aim 2 is to elucidate the structure of the RSV Gag lattice. With collaborators we will attempt to determine the structure of the SP assembly domain in the Gag lattice and also as a peptide, and attempt to determine how dimerization triggers Gag assembly in vitro. We will follow up a crystallization lead for the minimal Gag fragment needed for assembly. Finally, with collaborators we will determine the overall shape of RSV Gag, both in solution and when bound to tethered membranes.
Though the HIV/AIDS epidemic has been slowed, there is continuing need for drugs that target different steps in the virus life cycle. Virus assembly is a promising target but is incompletely understood. This proposal seeks to expand our understanding of two fundamental steps in the retroviral replication cycle, interaction of the vira structural protein (Gag) with the plasma membrane, and assembly of the Gag lattice that comprises the core of the immature virus particle.
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