Key steps of early HIV-1 infection include reverse transcription, nuclear import of replication complexes and transport to nuclear speckles followed by viral DNA integration into host genome. At some point before integration, the capsid shell surrounding the viral ribonucleoprotein complex must disassemble (uncoat) to release the viral pre-integration complexes. It is currently unclear where in the cell uncoating occurs and whether the capsid (CA) protein is progressively or synchronously lost from the capsid shell, yet optimal core stability is essential for evading the host innate immune responses and for nuclear import of functional viral complexes. Highly divergent findings regarding HIV-1 uncoating have been reported by several groups based upon the visualization of single virus uncoating in live cells. These conflicting results stem, in part, from the use of indirect CA labeling approaches and lack of minimally invasive direct fluorescent labeling of HIV-1 capsid. We hypothesize that HIV-1 uncoating is a multi-step process that involves permeabilization of the capsid shell in the cytoplasm, remodeling at the nuclear pore, and loss of CA in the nucleus. We will use a novel minimally invasive direct CA labeling strategy, which is based on site-directed incorporation of non-canonical amino acids and click- labeling with an organic dye, to elucidate single HIV-1 core permeabilization and uncoating events resulting to infection (Aim 1). Another gap in knowledge pertains to post-uncoating processes leading to HIV-1 integration and sub-nuclear compartments where integration occurs. We hypothesize that the viral pre-integration complex separates from the capsid shell and travels to the edge of a nuclear speckle where it engages the integrase- binding host factor LEDGF/p75 for integration into host genome. We will employ a novel live-cell single viral DNA visualization technology to track nuclear transport and productive integration of single viral complexes that establish actively transcribing viral RNA foci (Aim 2). Finally, we will use a powerful panel of biochemical, biophysical, structural biology, virology, and microscopy techniques to characterize a novel HIV-1 CA binding host factor, RBM14, which we hypothesize to modulate pre-integration steps of infection after nuclear import (Aim 3). These experiments are expected to elucidate the controversial HIV-1 uncoating process, reveal the dynamic events leading to productive integration and sites of integration, as well as delineate the role of RBM14- capsid interactions in early infection, thus informing novel antiviral strategies.