HIV-1 uncoating location dictates sites of integration.

HIV-1 cores enter the nucleus and undergo capsid disassembly (uncoating) near their integration site. Although most viral cores are localized to nuclear speckles (NSs), the spatial relationship between the uncoating site and integration site remains unclear. Here, using fluorescently labeled HIV-1 cores and NS markers, we show that uncoating predominantly occurs within NSs. Treatment of infected cells with capsid inhibitors PF-3450074 (PF74) or lenacapavir (LEN) after nuclear entry induced rapid disruption of interactions between capsid and cleavage and polyadenylation specificity factor 6 (CPSF6) followed by exit of HIV-1 cores from NSs, indicating that CPSF6 binding is required to retain the viral cores in the NSs. Treatment with PF74 or LEN led to core disruption and appearance of transcriptionally active proviruses further from the NSs compared to viral cores that uncoated in the NSs in untreated cells. This spatial shift correlated with reduction in integration into gene-rich, transcriptionally active speckle-associated chromatin domains, the preferred sites of HIV-1 integration, and increased integration into gene-sparse lamina-associated domains located away from the nuclear envelope. These findings demonstrate that the HIV-1 uncoating site is a key determinant of integration targeting, and that capsid inhibitors can misdirect integration by relocalizing uncoating to outside of NSs.