PROJECT SUMMARY HIV continues to be a major burden to global health as it accounts for approximately 36.9 million people living with acquired immunodeficiency syndrome (AIDS). Scientists have made considerable strides in understanding the basic molecular biology of HIV, which directly led to the development of effective antiretroviral therapy (ART). Yet, despite years of research, we currently do not have a complete picture of the HIV lifecycle to help develop a functional cure. By understanding key steps in the HIV lifecycle and establishing the cellular consequences of HIV infection, we can develop novel treatments and a functional cure, which take advantage of the cellular markers that HIV infection leaves behind. One key aspect of HIV biology that remains unclear is Vpr, an accessory gene without a known primary conserved function. Vpr is evolutionarily conserved among extant primate lentiviruses and is required for virus replication in vivo. Although Vpr enhances viral replication in monocyte-derived macrophages and monocyte-derived dendritic cells, it remains unclear how Vpr enhances replication. An emerging phenotype described for Vpr is engagement with the DNA damage response (DDR). The DDR is a signaling cascade responsible for safeguarding the genome from genotoxic assault that is well understood. Modulation of the DDR is often seen by diverse RNA and DNA viruses to promote tumorigenesis or enhancement of viral replication. Most interestingly, we found that Vpr can induce DNA damage and the ability of Vpr to induce DNA damage is conserved among HIV-1 and HIV-2. In addition, activation of the DDR is linked to NF-kB and innate immunity. Our preliminary data suggest that Vpr-induced DNA damage activates NF-kB. This data contributes to our central hypothesis that Vpr-induced DNA damage signals through the classical DDR to activate NF-kB target genes and enhance viral replication. In this proposal, we will investigate how and why Vpr induces DNA damage. In Aim 1, I will characterize the requirements and downstream signaling of Vpr-induced DNA damage. Completing this aim will shed light on how Vpr induces DNA damage and establishes if the classical DDR is activated or if non-classical activation mechanisms await to be explored. In Aim 2, I will identify the role of Vpr-induced DNA damage activation of NF- κB target genes in HIV replication. Completing this aim will determine why Vpr causes DNA damage and will establish the molecular mechanism for Vpr-enhancement of viral replication. Overall, we will gain insight into how HIV infected cells are dealing with DNA damage. The long-term goal is to leverage our understanding of DNA damage in HIV infected cells to develop improved treatments and a functional cure for people with HIV/AIDS.