SUMMARY HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in the nucleus during infection, we demonstrated that higher-order capsid complexes and/or complete cores containing the viral genome are imported into the nucleus. Inhibition of RT does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. Cytosolic and nuclear fractions of infected cells reveal that most RT intermediates are enriched in nuclear fractions, suggesting that HIV-1 RT occurs in the nucleus alongside uncoating. In agreement, we find that capsid in the nucleus induces recruitment of cleavage and polyadenylation specific factor 6 (CPSF6) to SC35 nuclear speckles, which are highly active transcription sites, suggesting that CPSF6 through capsid is recruiting viral complexes to SC35 speckles for the occurrence of RT. Thus, nuclear import precedes RT and uncoating, which fundamentally changes our understanding of HIV-1 infection.

Highlights d Depletion of cyclophilin A (CypA) expression in CD4 + T cells blocks HIV-1 infection d CypA binding to the core prevents HIV-1 restriction by TRIM5a during T cell infection d Endogenously expressed TRIM5a binds to the HIV-1 core and blocks reverse transcription d Human TRIM5a binds to HIV-1 cores but not to cores bearing capsid changes A92E/G94D

SERINC5(S5) is a multi-span transmembrane protein that potently blocks the infectivity of HIV-1 produced by human T-cells. The ability of S5 to restrict infectivity correlates with its presence in the virion, but the exact mechanism by which S5 restricts HIV-1 is unknown. Here we tested whether the core from HIV-1 virions containing S5 is delivered to the cytoplasm. Using the "fate of the capsid" assay, we demonstrated that the viral core of S5-restricted HIV-1 does not reach the cytoplasm of target cells, suggesting a block in the delivery of the core to the cytoplasm. In agreement with evidence suggesting that the viral determinants for S5 restriction map to the envelope of HIV-1, we observed that S5 induces conformational changes to the HIV-1 envelope. Further, we demonstrated that S5 localizes to detergent-resistant membranes (DRMs), as has been shown previously for the HIV-1 envelope in producer cells. In order to identify the determinants of S5 restriction, we explored the ability of all human SERINC proteins to restrict HIV-1. In contrast to human S5, we observed that human SERINC2(S2) did not restrict HIV-1, and was inefficiently incorporated into HIV-1 virions when compared to S5. Experiments using S5-S2 chimeric proteins revealed two functional domains for restriction: one necessary for S5 incorporation into virions, which does not seem to be necessary for restriction, and a second one necessary to change the HIV-1 envelope conformation, localize to DRMs, and block infection.

HIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

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