Background
The nuclear import of HIV-1 into the host nucleus marks a milestone of the viral infection, making it a potential drug target for effective prophylaxis and treatment. However, understanding this process has been challenged by its dynamic nature and the scarcity of HIV-1 cores in infected cells.
Methods
To elucidate the mechanisms underlying the translocation of HIV-1 capsids into the nucleus and their interactions with nuclear pore complexes (NPCs), we developed a robust system that recapitulates the nuclear import of HIV-1 cores into intact nuclei using mechanically lysed or detergent permeabilized T cells with purified HIV-1 cores. We employed cryo-focused ion beam (cryo-FIB) milling and planar lift-out, guided by cryogenic correlative fluorescence light and electron microscopy (cryo-CLEM), to prepare thin lamellae of T-cell nuclei containing HIV-1 cores. These developments enabled the following correlative cryo-electron tomography (cryo-ET) analysis of HIV-1 core nuclear import in situ.
Results
By utilizing these technologies, we achieved a productive capture of HIV-1 cores at different stages of nuclear import, with an unprecedented correlation efficiency of over 52%. Among the 510 HIV-1 cores identified in our tomograms, 87% of cores were specifically associated with nuclei. Statistical analyses of core morphology and size revealed, intriguingly, that tube-shaped and small HIV-1 cores were more advantageous in nuclear import compared to larger cone-shaped cores. Moreover, some large HIV-1 cores appeared to undergo deformation during the passage. Interestingly, NPCs, on the other end, were observed to dilate and occasionally crack, upon the entry of HIV-1 cores. We further observed the surface of capsid is coated with distinct densities, potentially corresponding to FG meshes, when passing through the NPCs.
Conclusions
This work introduces a robust in situ system with a highly efficient correlative cryo-ET workflow and provides novel insights into the nuclear import of HIV-1, which not only advances HIV-1 research but also opens a new avenue for studies on nuclear import mechanisms.
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