Importin 7 mediates the nuclear import of HIV-1 integrase via a specific interacting interface.

HIV-1 integrase (IN) must cross the nuclear envelope to access the host genome and catalyze viral DNA integration, a process that requires active nuclear import. Although recent work has established that the primary pathway for HIV-1 nuclear entry involves transport of the intact capsid, complementary mechanisms may enable the nuclear import of individual viral components through the host karyopherins. Among the host nuclear import factors implicated in this process, importin 7 (Imp7) has emerged as a strong candidate for IN, yet its precise role and the molecular basis of its interaction with IN have remained unclear. Here, we demonstrate that Imp7 acts as a critical mediator of IN nuclear import. Using hydrogen-deuterium exchange and cross-linking mass spectrometry, we map the interaction interface and identify the core domain of Imp7 as the primary IN binding site. Affinity measurements reveal high-affinity binding between IN and Imp7, while mutations within the C-terminal nuclear localization signal of IN significantly weaken this interaction. Consistent with these findings, knockdown of endogenous Imp7 in HEK293T cells leads to cytoplasmic accumulation of IN, confirming its essential role in nuclear import. Together, these results establish a direct, high-affinity interaction between IN and Imp7, define their molecular interface, and position Imp7 as a key nuclear import receptor for HIV-1 IN. This work provides detailed molecular insight into a critical host-virus interaction during early HIV-1 replication and highlights the Imp7-IN interface as a promising target for complementary therapeutic intervention aimed at disrupting integrase nuclear import.