Cryoelectron tomography of HIV-1

University Of Utah, Salt Lake City UT

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Abstract

Cryoelectron tomography of HIV-1 virus-like particles (VLPs) at multiple stages from budding to maturity is proposed. This work capitalizes an two recent advances: (1) the development of electron microscopes and techniques that can record "molecular resolution" (-4nm), three-dimensional tomograms of unique objects the size of HIV-1, and (2) the creation of non-infectious but structurally authentic virus-like-particles (VLPs) that can be studied safely in a native, frozen-hydrated state (without fixation or embedment) in the laboratories that house these microscopes. Tomograms will be recorded of fully mature VLPs, VLPs arrested at different stages of maturation by mutation of specific protease sites within the Gag protein, immature VLPs lacking functional protease, and VLPs arrested in a late-stage of budding within host-cell membranes. These tomograms should be of sufficient resolution to (1) test the existence of the core-envelope linkage (CEL) structure, (2) rigorously measure the cone angle of the viral core to test the "fullerene cone" model for capsid structure, (3) define the positions of various viral components, including cyclophilin A and the supramolecular replication initiation/RNA parkaging/dimer linkage structure, (4) visualize the dynamic processes of budding and maturation, and (5) test molecular models for the organization of the matrix, capsid, nucleocapsid, and Gag shells of mature and immature HIV. Once the arrangement of the repeating units in these structural shells is determined, subunits will be classified according to position and averaged to improve resolution and guide the docking of known atomic models. This work will accelerate the development of HIV assembly inhibitors by revealing the number and nature of protein contacts and conformations involved in the HIV-1 life cycle and by providing the means to characterize how such inhibitors are acting.