Single-particle Reconstruction of HIV-1 Envelope Glycoprotein Trimers

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DESCRIPTION (provided by applicant): The availability of detailed structures of biomolecules can expedite the translation of genomic and proteomic information into therapeutic and prophylactic interventions. Unfortunately, the size, pleiomorphism and/or conformational flexibility of some proteins renders them refractory to conventional approaches to obtaining structure, such as x-ray crystallography or NMR spectroscopy. One such example is the trimeric human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein complex, which mediates the entry of the virus into the host cell. The unliganded HIV-1 Env glycoprotein complex exists in a high-energy state; upon binding to the CD4 and CCR5 receptors, the HIV-1 Env glycoproteins assume lower-energy conformations. These conformational transitions in the HIV-1 Env glycoprotein complex ultimately result in the fusion of the viral and target cell membranes. As the only virus-specific protein exposed on the HIV-1 membrane, the Env glycoprotein trimer represents a major target for entry inhibitors, including small molecules, peptides and neutralizing antibodies. Unfortunately, conformational flexibility, the lability of the unliganded state, and a high degree of glycosylation have slowed structural studies of the Env glycoprotein trimer. This gap in knowledge represents a major barrier to progress in HIV-1 entry inhibition and vaccine immunogen design. The proposed studies will utilize single-particle cryoelectron microscopy to yield structures of membrane-anchored HIV-1 Env trimers in different conformations at near-atomic (3.5 - 5 E) resolution. The Specific Aims of this application are: 1) To prepare membrane-anchored HIV-1 Env glycoprotein trimers that are suitable for high-resolution structure determination by single-particle cryoelectron microscopy and to solve the structure of the unliganded HIV-1 Env trimer; 2) To investigate the structure of the HIV-1 gp41 cytoplasmic tail and its potential contribution to the structure of the Env ectodomain; and 3) To solve the structure of the CD4-bound conformation of the HIV-1 Env glycoprotein trimer. These studies will yield detailed snapshots of two key stages in the process of HIV-1 entry, providing a framework for understanding the dynamic aspects of the Env glycoprotein "membrane-fusing machine". Conserved, functionally important structures on the HIV-1 Env glycoprotein complex that can serve as targets for drugs or vaccine-induced antibodies will be revealed. This information will transform our understanding of HIV-1 entry and should inspire new approaches to intervention.