PROJECT SUMMARY To infect a target cell, the Human Immunodeficiency Virus-1 (HIV-1) must complete a series of steps initiating with fusion and release of the viral core into the cytoplasm, followed by reverse transcription, nuclear translocation, and integration into the host genome. The intermolecular relationships between core components and host proteins during intracellular transport to the nucleus are likely incompletely defined. Identifying and understanding such interactions is important for defining viral cofactors and innate host defenses and has implications for the generation of novel therapeutics. This proposal aims to utilize advances in the fields of high-resolution imaging and proteomics to develop methods capable of providing new insights into HIV-host protein interactions during the early stages of infection. The identification of cellular cofactors necessary for productive HIV infection and of the cellular proteins that provide innate defenses is likely hampered by the weak and temporal affinities of viral and cellular proteins for one another. Recently developed proximity-labeling technologies present a novel approach to overcome such limitations, by using enzymatic activities that can generate free biotinyl radicals to enable rapid and spatially-restricted labeling of proteins proximal to the enzyme. This enables weak interactions to be identified, which would otherwise be lost through standard affinity purification. A key advantage of this approach is that enzyme-mediated proximity labeling can also be imaged by electron microscopy. We hypothesize that fusion tagging of virion proteins with the enzyme ascorbate peroxidase (APEX) will ultimately enable us to define the proximity-proteome of virion cores and preintegration complexes in target cells and correlate this with subcellular localization. Here, we propose to construct and characterize fusion proteins of capsid and integrase, toward the long term goal of combining high-resolution imaging and proteomics to define the itinerary and interactions of HIV-1 core components during the early stages of infection. We also propose to optimize a protocol for using these new constructs to resolve the early events of HIV-1 infection at the ultrastructural and proteomic levels. We will first use an already-generated fusion of APEX2-Vpr as a prototype for establishing the experimental conditions necessary for the detection of viral cores and proximally-labeled proteins by electron microscopy and quantitative mass spectrometry. Using these conditions, we will extend our study to track the localization and proximity-proteome of the viral components integrase and capsid, using our new fusions of APEX2 to these proteins. Should these methods prove successful, we will ultimately validate novel cellular interactors identified by our proximity labeling proteomics studies, demonstrating the utility of APEX2 as a discovery pipeline that can subsequently be applied to other HIV proteins and targets.