Structural interrogation of the HIV-1 virion and the mechanism of the capsid targeting compound Lenacapavir

Central to HIV-1 assembly, maturation, and entry are two structurally distinct protein lattices formed by the viral protein Gag. The immature lattice is formed by the uncleaved Gag protein. Maturation is brought about by retroviral protease cleavage of Gag, and subsequent assembly of the liberated CA domain into a mature lattice that encloses the genome and viral enzymes like RT and IN (the Capsid Core). Following fusion and release of a Capsid Core into the cell cytoplasm, a series of interactions with cellular proteins can result in “docking” to a nuclear pore. Premature disruption of the CA lattice can restrict infection. A structural understanding of these states and transitions is key to the design and optimization of antivirals that target capsid. The work proposed here will leverage single-particle cryo-Electron Microscopy and cryo-Electron Tomography and Subtomogram Averaging to interrogate the structural details of immature HIV-1 assembly, and the mechanistic properties of mature Capsid. This work will employ continued development and application of novel sample preparation for cryo-EM, and data processing approaches to determine the tertiary and quaternary structure of these assemblies. Samples biochemically assembled in vitro from purified proteins will be studied to gain a precise understanding of the molecular determinants of these processes. Virions from cells will be examined to understand how these molecular determinants act in the native virus. Cells will be used to investigate how these actions relate to the cellular environment. Time-resolved sample preparation will be applied to samples at specific initiation states of assembly, which may represent novel targets for inhibition. Specific Aim 1: DEVELOPMENT OF SINGLE PARTICEL CRYO-EM TO STUDY THE IMMATURE AND MATURE GAG AND CAPSID LATTICES. Assembly and budding of HIV-1 is a tightly regulated process involving protein-protein, -RNA, -membrane, and -cellular cofactor interactions. A more detailed understanding of the initiation of assembly and the transition to a budded virus may reveal targets to block the release of the virus. Application of cryo-EM to investigate these dynamic processes has provided, and will continue to provide, important insights to HIV-1 biology. My lab will work to be a leader in this area by developing new and novel approaches to sample preparation and structure determination as outlined in this aim. Specific Aim 2: STRUCTURAL, BIOCHEMICAL AND CELLULAR INTERROGTOIN OF LEN BINDING TO HIV- 1. The mature Capsid Core contains all the necessary viral components for establishing a new infection. Following release into the cytoplasm, Capsid interacts with (or hijacks) a series of cellular proteins to facilitate trafficking to, and docking with, a nuclear pore. Compounds that bind to these capsid-cellular co-factor binding sites are an attractive and successful antiviral strategy. We will study the molecular details and effects of Lenacapavir, the first-in-class capsid targeting compound, on immature assembly, capsid stability, and infectivity. Project Number: 1R56AI189254-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Robert Dick | Institution: EMORY UNIVERSITY, ATLANTA, GA | Award Amount: $653,693 | Activity Code: R56 | Study Section: HIV Molecular Virology, Cell Biology, and Drug Development Study Section[HVCD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R56AI18925401