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

Abstract

<p class="first" id="P3">Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. HIV-1 and other lentiviruses are able to mediate the infection of non-dividing cells through the ability of the capsid (CA) protein ^{<a class="xref-link" href="#R1">1</a>} to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex (NPC) ^{<a class="xref-link" href="#R2">2</a>– <a class="xref-link" href="#R4">4</a>}. Although recent studies have observed the presence of capsid in the nucleus during infection ^{<a class="xref-link" href="#R5">5</a>– <a class="xref-link" href="#R8">8</a>}, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore 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 (&lt;5 hours) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription completes in the nucleus. We also observe that HIV-1 remains susceptible to a capsid destabilizing compound PF74 following nuclear import, revealing that uncoating completes in the nucleus. We also observe that certain CA mutants are insensitive to a Nup62 mediated nuclear pore blockade in cells which potently block infection by wild type CA, demonstrating that HIV-1 can utilize distinct nuclear import pathways during infection. These studies collectively define the spatiotemporal 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. </p>