HIV-1 and SIV Envelope Glycoproteins

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DESCRIPTION (provided by applicant): Detailed knowledge of the structural and functional aspects of the molecules involved in the interaction, binding, and fusion of HIV-1 and the closely related SIV with their target cells is fundamental to our understanding of the disease processes in AIDS and to the rational design of diagnostic reagents, vaccines and other therapeutic agents. Two of the most important molecules in the infectious process, namely the envelope glycoproteins (Env) glycoproteins, SU (surface), and TM (transmembrane), are only partially characterized at the structural level with many of the important features are still poorly defined. These areas of uncertainty include the orientation of the variable loops, the structural changes that occur during the binding and fusion processes, and the features which foster or inhibit Ab-mediated neutralization. Another area of uncertainty involves the manner by which the Env proteins are expressed on the surface of virus particles. There is, for example, controversy regarding the number of Env spikes on the viral surface, their distribution and their stability. This proposal describes investigations aimed at applying new approaches for the determination of the 3-D morphology of the HIV-1 and SIV Env, gp120 and gp41, as expressed on the viral surface and in purified recombinant form. Our goals are to determine the numbers, locations, orientations, and structural details of Env molecules, both alone and in complex with monoclonal antibodies and ligands, on the surface of various forms of HIV-1 and SIV viral particles. In addition we will expand upon our previous epitope and reactive-site mapping studies of individual molecules and complexes by conducting 3-D structural analyses on 2-D crystals composed of purified monomeric and trimeric Env proteins and immune complexes. The primary methodologies will be the analysis of intact virions by negative stain and cryo-electron microscopic (EM) tomography and the analysis of purified recombinant Env and immune complexes by electron diffraction of 2-D crystalline arrays.