HIV/AIDS is a disease that affects millions of people worldwide. To develop effective HIV-1 therapies it is important to understand the fundamental biology behind the steps in infection of target cells. The studies outlined here will further elucidate the uncoating step of the viral replication cycle, a process which has yet to be well defined and is not directly targeted by current HIV-1 therapies. HIV replication begins when a virion fuses with the target cell membrane, resulting in the release of a viral complex into the cytoplasm of the cell. This viral complex consists of the HIV-1 genomic RNAs and associated proteins surrounded by a conical shell of p24CA protein. In order for infection to progress, p24CA must dissociate from the rest of the viral complex, a process referred to as uncoating. Although uncoating is required for HIV-1 replication, many questions remain as to the timing and factors involved in this process. During early steps of infection, the viral RNA genome is copied through a process called reverse transcription into DNA which is then integrated into the chromosomal DNA of the target cell. Recent studies suggest that there is a complex interplay between the process of uncoating and reverse transcription. Therefore, the goal of this proposal is to further elucidate the role of reverse transcription in HIV-1 uncoating. The proposed experiments will test the hypothesis that early steps in reverse transcription are necessary to facilitate initiation of uncoating. Three novel assays will be used to study uncoating in HIV-1 infected cells. The CsA washout assay uses a protein from owl monkeys, TRIM-CypA, to inhibit infection of HIV-1 viral complexes in cells. The other two assays use microscopy to visualize fluorescently labeled HIV-1 virions in cells. In the in situ uncoating assay populations of virions are examined at various time points to determine the rate of uncoating. In the live cell uncoating assay the uncoating of individual virions in living cells is examined in real time. These experiments will be complemented using molecular biology techniques to characterize reverse transcription. The experiments proposed in Aim 1 will determine if reverse transcription facilitates initiation or progression of uncoating by studying p24CA mutants and the effect of reverse transcriptase inhibitors on uncoating. In Aim 2 the process of reverse transcription will be characterized with respect to the timing of uncoating in infected cells. Mutations that disrupt reverse transcription will also be used to determine if progression of reverse transcription to a specific intermediate DNA product is necessary to facilitate uncoating. Finally, uncoating has not been characterized in real time in infected cells. In Aim 3 the live cell uncoating assay will be used to determine the kinetics of uncoating and how mutations in p24CA and reverse transcription alter this process. In summary, these studies seek to provide a better understanding of the process of uncoating and the interplay between uncoating and reverse transcription.