Dissecting HIV Capsid Uncoating using Advanced Imaging and Cryo-Electron Tomography - PROJECT SUMMARY Upon entry into target host cells, HIV releases its conical capsid (CA) core into the cytoplasm, which contains viral genes and enzymes that are necessary for infection. The core must disassemble, or “uncoat”, as it traffics to the genome – a process that is crucial for exposing the viral DNA for host chromatin engagement. Uncoating must be tightly regulated, because i) the virus must effectively deliver the viral genome to host chromatin while ii) protecting its genome from host DNA sensing systems. Disruptions to CA morphology lead to ineffective uncoating and reduced infectivity. Small molecule inhibitors, such as Lenacapavir (LEN), block uncoating and inhibit infection by disrupting CA morphology. Thus, both the stability of the core and the regulation of uncoating are critical for successful infection. Although the functional role of CA is understood, the mechanism and location of core uncoating, and how small molecule inhibitors disrupt core morphology, remain poorly understood. This project has two specific aims that will address major gaps in the current understanding. Aim 1 focuses on dissecting the structural basis of HIV uncoating in vitro. This will be accomplished by employing a novel in vitro uncoating assay coupled with correlative light and electron microscopy (CLEM), cryo-electron tomography (cryo-ET), and image analysis to identify, image, reconstruct, and interpret the morphologies of CA cores, and how they change during the uncoating process. Extending from these data, the project will also define how small molecule inhibitors, such as LEN, affect CA core morphologies. Aim 2 will capture and image HIV uncoating inside cells to provide insights of this fundamental process in its native state and during cellular infection. In brief, cells infected with HIV will be vitrified at multiple timepoints after infection. Electron transparent samples will be prepared using focused-ion-beam milling, which will be coupled with CLEM and cryo-ET for data collection. Subsequently, high-resolution images will be analyzed to gain insights into CA core morphologies in situ, their cellular locations, and how cores change as a function of space and time. Collectively, these data will elucidate the mechanisms of CA core uncoating, assess the impact of small molecule inhibitors on core stability, and interrogate spatiotemporally how and where uncoating occurs in cells. This research proposal will create an interdisciplinary training environment for applicant Ms. Rodriguez. In addition to having a various team of mentors, Ms. Rodriguez will have a network of collaborators to expand her technical, computational, and analytical skills. Ms. Rodriguez will develop technical skills, including culturing cells, infecting cells with virus, CLEM, FIB-milling, cryo-ET sample preparation, data collection, and processing. The candidate will attend professional development workshops, conferences, and seminars to further develop her science communication skills, and expand her professional network. Additionally, Ms. Rodriguez proposes to mentor undergraduates in the laboratory and teach courses. This will enable her to also develop as a mentor herself. The support of this fellowship will help the applicant develop into an independent researcher.
