ABSTRACT, PROJECT 2 After infecting a cell, HIV reverse transcribes its RNA into viral DNA (vDNA) that integrates into the host cell genome. The vDNA must enter the nucleus to access the host DNA for integration, but how the vDNA gains nuclear entry, migrates to sites of integration, and selects sites of integration are essential yet poorly understood steps in HIV replication. A thorough understanding of how the virus exploits host factors to complete these key steps in replication is needed to identify promising molecular targets for development of new antiretroviral therapies. Following nuclear import, intact viral cores are transported to interchromatin domains called nuclear speckles where they uncoat so that the preintegration complex (PIC) can integrate the vDNA into nearby chromatin. In Aim 1, we will identify and elucidate dynamic HIV core-host cell interactions that influence nuclear uncoating efficiency/kinetics and the nuclear penetration distance of viral cores; we will also determine whether the HIV core-host interactions affect genome-wide selection of integration sites using multiple synergistic experimental approaches. The main substrate for HIV integration is chromatin and nucleosomal DNA, and integration into transcriptionally active genes is favored by association of integrase (IN) with lens epithelium derived growth factor (LEDGF). How HIV PICs interact with LEDGF and nucleosomes and which target sites are favored for integration by purified assembled IN-vDNA complexes (intasomes) has not yet been determined. In Aim 2, we will construct physiologically relevant chromatin targets of integration in vitro, define preferred regions for viral integration into nucleosome substrates and elucidate how host factors and chromatin states modulate integration site preferences. These directions will elucidate the mechanistic bases by which viral/host interactions direct viral DNA integration into chromatin. It is generally thought that the chromatin state at integration sites determines transcriptional activity or progression to latency. However, host chromatin states and viral factors that control establishment of active or latent infection remain enigmatic. As HIV latent reservoirs reinitiate infection upon cessation of antiretroviral therapy, they have thus far prevented development of a cure for HIV infection. We will develop multiple experimental approaches to elucidate mechanisms that establish latent infection and reinitiate transcription, which may promote development of latency-reactivating or latency promoting treatments and achieve eradication or a functional cure for HIV infection. In Aim 3, We will develop multiple experimental systems to determine the roles of IN, Tat and in vivo integration sites to examine the role of the integration site chromatin in establishment of active vs. latent infection. Overall, these studies will provide transformative insights into HIV uncoating, integration site selection, the molecular machine...