Time-Resolved Fluorescence Imaging and Correlative Cryo-Electron Tomography to Study Structural Changes of the HIV-1 Capsid.

The conical HIV-1 capsid protects the internal viral genome and facilitates the infection of target cells. Highly potent antivirals, such as the clinically approved drug Lenacapavir (LEN), block HIV-1 replication by changing the capsid structure and modulating its function. However, structural studies of the HIV-1 capsid, its disassembly, or stabilization by antivirals have been challenging. Here, we developed a correlative light and cryo-electron microscopy (CLEM) workflow to characterize HIV-1 capsid morphology, starting from a small volume of viral particles harvested from cellular supernatants. We report two critical improvements in sample preparation, namely, (1) affinity capture and retention of fluorescent HIV-1 particles on cryo-EM grids to enable mapping virus/capsid location prior to sample vitrification and (2) streamlined alignment protocols to subsequently identify and correlate regions of interest in fluorescence and cryo-EM images. These improvements enable a reproducible CLEM workflow to accurately locate capsids for cryo-electron tomography (cryo-ET) studies. Using this approach, we resolved ultrastructures of HIV-1 capsids treated with LEN and the cellular metabolite inositol hexaphosphate (IP6), revealing distinct modes of capsid lattice stabilization. Finally, using our CLEM workflow, we demonstrate the feasibility of correlating time-resolved fluorescence imaging of capsid disassembly to end point cryo-ET structures. These advances will facilitate in vitro structural studies to define the mechanisms of HIV-1 capsid stabilization and disassembly. The CLEM workflow developed here can also be extended to studying structural changes in other viruses in response to diverse stimuli.