Correlative In Situ Cryo-ET Reveals Cellular and Viral Remodeling Associated with Selective HIV-1 Core Nuclear Import

Lentiviruses like HIV-1 infect non-dividing cells by traversing the nuclear pore, but studying this process has been challenging due to its scarcity and dynamic nature in infected cells. Here, we developed a robust cell-permeabilization system that recapitulates HIV-1 nuclear import and established an integrated cryo-correlative workflow combining cryo-CLEM, cryo-FIB, and cryo-ET for targeted imaging of this process. These advancements enabled the successful capture of 1,899 HIV-1 cores at various stages of nuclear import. Statistical and structural analyses of native wild-type and mutant cores revealed that HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability, as well as nuclear factors such as CPSF6. Brittle cores fail to enter the nuclear pore complex (NPC), while CPSF6-binding-deficient cores stall inside the NPC, resulting in impaired nuclear import. Intriguingly, nuclear pores function as selective filters favoring the import of smaller, tube-shaped cores. Our study opens new avenues for dissecting the biochemistry and structural biology of HIV-1 nuclear import as well as downstream events including core uncoating and potentially integration, with unprecedented detail.