HIV-1 enters the nucleus of non-dividing cells where the reverse transcribed viral DNA is integrated into the host genome. Whereas the nuclear pore complex (NPC) prevents passive transport of large macromolecules, HIV-1 has evolved effective strategies to penetrate this barrier. The HIV-1 nuclear import is a poorly understood process that involves complex interactions with the nuclear import machinery, including several nucleoporins, transportin-3, and CPSF6, all of which bind the viral capsid core, which comprises hundreds of copies of the capsid protein (CA). Pleiotropic effects caused by nucleoporin knockdown and the ability of HIV-1 to use alternative import pathways have impeded the mechanistic studies of nuclear import and frustrated efforts to identify the full array of host factors involved in this process. Our single particle tracking experiments revealed that HIV-1 infection progresses through CA-dependent docking at the nuclear membrane, followed by uncoating (loss of CA) and CA-dependent nuclear transport to the sites of integration. However, very little is known regarding the molecular details and dynamics of virus-NPC interactions, including the structural changes in the architecture of both HIV-1 and the NPC in the course of nuclear import. There is thus an unmet need for structural and functional studies on the molecular mechanisms of HIV-1 nuclear import in the context of productive infection. We propose to combine cutting-edge approaches developed by our highly collaborative team to delineate the molecular interactions and structural changes in the virus and NPC during the nuclear import. Specifically, we will: (1) identify the host factors involved in HIV-1 import using novel proteomics and chemical cross-linking approaches; (2) obtain cryo-electron tomography (cryo-ET) and cross-linking mass-spec structures of the HIV-1 core/NPC complexes by capturing the incoming virus through our new on-demand pore clogging assay; and (3) develop correlative fluorescence/cryo-ET imaging pipeline to structurally characterize the intermediates of HIV-1 nuclear import. Knowledge of molecular interactions during the HIV-1 nuclear import should help identify novel therapeutic targets to block infection, provide a framework for studies of the nuclear import of other viruses and further fundamental understanding of the nuclear pore function.