Project Summary The HIV pandemic has infected 72 million people worldwide, and in the US, an estimated 1.2 million people are currently living with HIV. Despite considerable progress, antiretroviral therapy (ART) remains the only viable treatment option to curb the disease burden. However, the current ART regimens are toxic, costly, and face viral resistance; beckoning the need for novel antiretroviral targets. A promising ART target is HIV integration, the key step for infection. HIV-1 integration targets actively transcribing regions of the host genome for integrating the viral DNA and establishing infection. Recent reports suggest that the targeting of actively transcribing genes is associated with clonal expansion and the subsequent viral latency in patients, a primary barrier for a cure. It is well established that actively transcribing genes are characterized by an ?open? chromatin state, in contrast to a ?closed? state indicative of suppressed regions of the genome. Post- translational modifications (PTM) of the histones define these open and closed regions of the genome, and the resulting genomic function. In a recent report, HIV-1 hotspots were shown to align with PTM associated with active transcription. A major question is how HIV targets these open regions of the genome? In cells, HIV integration is carried out by a nucleoprotein complex of viral DNA and viral/host proteins, called the preintegration complex (PIC). The viral proteins in the PIC hijack host proteins for the fitness of the HIV infection. However, PIC function in the context of chromatin, the natural target of HIV-1 integration, and specific histone PTM is understudied. Our lab has established a protocol to isolate and study intact, naturally forming PICs. This novel tool allows us to investigate the specific activity of the HIV-1 PIC and define the factors that are important to integration. The central hypothesis of the proposed studies is that chromatin characteristic of active transcription increases HIV-1 PIC activity. These studies will address the role of chromatin in HIV integration by (1) defining the preferred DNA substrate for HIV-1 PIC activity. Then using biochemical and cellular approaches we will (2) probe the mechanism of integration into chromatin. The knowledge gained from this investigation will aid in the development of novel drugs against HIV infection.