Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus

Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein¹ to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex^2-4. Although recent studies have observed the presence of the capsid protein in the nucleus during infection^5-8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.