BPS2026 – Visualizing HIV-1 capsid structural changes through time-resolved fluorescence microscopy and correlative cryo-electron tomography

Fusion of the viral and cellular membranes releases the HIV-1 conical capsid (CA) core into the target cell. Capsid disassembly, or “uncoating,” is a prerequisite to successful genomic integration of the viral DNA into host chromatin. However, the molecular mechanisms of uncoating, and the accompanying morphological changes in the CA core, remain unknown. Using time-lapse fluorescence microscopy and cryo-electron tomography (cryo-ET), we developed a correlative light electron microscopy (CLEM) workflow to study HIV-1 core uncoating in vitro. Fluorescently tagged HIV-1 virions are adhered to grids, and saponin is added to strip the viral membrane and initiate the capsid uncoating process. Treatment of permeabilized cores with small molecules, such as IP 6 and Lenacapavir (LEN), allowed us to study their respective effects on capsid morphology, by cryo-ET. Fluorescence images that were collected before vitrification were used to enable identification of HIV-1 cores by CLEM under the TEM, for subsequent cryo-ET tilt series data collection, 3D reconstructions and structure determination. Our results indicate distinct core morphologies between untreated virions and those treated with small molecule inhibitors, such as IP6 and LEN. We present the results demonstrating the utility of our CLEM workflow that allows us to correlate virions and small-molecule treated cores from fluorescence microscopy to cryo-ET and image reconstruction. These in vitro assays will facilitate differentiating intact from partially assembled CA cores and elucidate structural changes during the uncoating process. An understanding of core morphologies will in turn enable identifying distinct types of core assemblies in situ.