PROJECT SUMMARY To initiate an infection, HIV-1 must enter the cell, reverse transcribe the viral RNA into DNA, enter the nucleus, and integrate into host cell chromatin. Studies in Project 1, Infecting the Cell, will focus on defining these key steps in the first half of the HIV-1 life cycle in molecular and mechanistic detail. A particular theme will be to elucidate the organization, functional roles, and dynamic transformations of the viral core particle. Studies in Aim 1 (Virus Entry) will build on our previous structural studies of different states of HIV-1 Env and imaging of viral entry complexes in situ to provide structural details of the virus-target cell interaction at the time of entry including: 1) the number of Envs and average spacing in a typical interaction with a target cell, 2) whether there is rearrangement of the Env subunits that are not bound to host receptors, and 3) the structure of the hypothesized pre-hairpin intermediate formed between the virus and host cell membranes by Env and host receptor(s) at sufficient resolution to dock coordinates from components of known structure. Studies in Aim 2 (Genome Structure and Reverse Transcription Initiation) will build on our previous structures of HIV-1 replication initiation complexes to reveal: 1) the structural basis for the transition from initiation to elongation, 2) how the dimeric structure of the viral RNA modulates reverse transcription initiation, 3) how the global dynamic architecture of HIV-1 genomic RNA is influenced by the capsid, and 4) how this dynamic architecture influences the process of reverse transcription, including strand jumps. Studies in Aim 3 (Capsid Functions in Infection) will build on our development of a cell-free system that efficiently reconstitutes the coupled processes of viral endogenous reverse transcription and integration to: 1) characterize the viral genomic template at different stages of reverse transcription, 2) reveal how the capsid facilitates reverse transcription, 3) define how the potent new capsid inhibitor, lenacapavir, binds the capsid and inhibits nuclear entry, and 4) reconstitute and image core passage through nuclear pores. Studies in Aim 4 (Core Uncoating and Integration) will build on our cell-free integration assay and our ability to reconstruct viral capsid lattices at high resolution to fill a series of fundamental knowledge gaps, including: 1) the identities and functional roles of host factors that promote uncoating and integration, 2) how the viral capsid “opens” to expose the integrating viral DNA, and 3) the 3D architectures of active, native intasomes.