Structural information about the HIV or SIV envelope glycoprotein will be important for design and development of vaccine immunogens. The overall goal of this component is to study the structure of the trimeric, """"""""pre-fusion"""""""" state of the HIV/SIV gp160 ectodomain (referred to as """"""""gp 140"""""""") and, in conjunction with the other components of the Project, to probe the immunological properties of gp140. The intial goal is to obtain by electron cryomicroscopy a moderate-resolution (12-18 A) image of trimeric SIV gpl40, chosen because of the greater stability of the trimer (with respect to HIV gp140). We will use single-particle analysis of individual gp140 trimers and of more symmetrical assemblies of trimers created by use of an ATCase fusion protein already successfully expressed. We will also attempt to make two-dimensional arrays on lipid substrates. We will determine the binding sites and orientations of neutralizing antibodies by """"""""decorating"""""""" gp140 trimers with Fabs and determining those structures by single-particle analysis, and we will determine the locations of variable loops through study of appropriate deletants. We will fit known substructures into the moderate resolution model, taking into account the positions of epitopes and variable loops, We will use the accumulated information to design a new generation of stablized, trimeric ectodomains in the prefusion conformation, for crystallization and structure determination by X-ray crystallography.
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