ABSTRACT This Project is designed to characterize the early post-entry steps of HIV-1 replication, core evolution, and TRIM5α restriction in structural and mechanistic detail. Following virus-cell fusion, the viral genomic RNA (gRNA) is reverse transcribed1-6, the core interacts with host factors and loses its capsid (“uncoating”)7-11, and the host restriction factor TRIM5α can recognize the capsid and block these transformations.12-16 To characterize these processes, we will determine 3D structures of replication initiation complexes that comprise reverse transcriptase (RT), the host tRNALys,3 primer, and various gRNA templates. We will study the conformational changes that accompany replication (Aim 1), develop and apply new single particle assays for studying reverse transcription within isolated viral cores (Aim 2), image transduced cells using fluorescent microscopy and correlated light and electron cryotomography (CLEM-ECT) to visualize and characterize core trafficking, reverse transcription, uncoating and restriction (Aim 3), and reconstitute, image and model TRIM5α- capsid complexes to learn how TRIM5α inactivates the core and stimulates innate immune responses (Aim 4).