PROJECT SUMMARY The negative strand of the HIV-1 proviral genome contains a highly conserved open reading frame (ORF) that overlaps the env gene straddling the gp120/gp41 boundary. This ORF encodes an antisense protein (ASP) of ~189 residues, rich in hydrophobic amino acids, and associated with cellular membranes. Expression of ASP is driven by a Tat-independent negative sense promoter (NSP) located in the 3’ long terminal repeat (LTR). The presence of humoral and cellular immune responses against the ASP protein in people living with HIV-1 (PLWH) provide evidence that ASP is expressed during HIV-1 infection in vivo. The ASP ORF is found exclusively in HIV-1 strains that belong to clades of the pandemic group M, and the percentage of strains in each clade that have an intact ASP ORF correlates with the worldwide prevalence of the clade. On the contrary, the ASP ORF is absent in all other human and non-human primate lentiviruses: the non-pandemic HIV-1 strains in groups O, N and P, HIV-2 strains, and SIV strains of all species. This suggests a possible accessory role of ASP in virus spread. Indeed, our studies have shown that ASP is expressed in the nucleus of non-productively infected cells, and on the cell surface of productively infected cells. In addition, upon viral budding and release, ASP is present on the envelope of cell-free HIV-1 virions. We also found that knocking out ASP expression reduces HIV-1 replication both in lymphoid and myeloid primary human cells. However, despite mounting evidence that ASP is expressed in vivo, and that it promotes viral replication, the function and the mechanism of action of ASP in HIV-1 infection and pathogenesis remain unknown. The elucidation of the role that HIV-1 proteins play in viral replication, in promoting HIV-associated pathologies, and in altering normal cell physiology has often relied on the development of transgenic (Tg) mouse models. The central hypothesis of this application is that development of an ASP Tg mouse model will facilitate the study of the function that ASP plays in the virus lifecycle, it will further advance our knowledge of HIV-1, and it may lead to novel therapeutic interventions. We propose two specific aims. Specific Aim 1 seeks to establish a Tg mouse model to study 3’LTR-driven, inducible expression of ASP in CD4+ cells, the natural targets of HIV-1 infection. We have developed a construct that will allow us to achieve those goals, and we have performed in vitro exper- iments to test its function. In addition to establishing the Tg model, this Specific Aim will confirm that ASP is expressed in CD4+ cells in vivo after induction. Specific Aim 2 seeks to identify the main phenotypic character- istics of ASP Tg mice. Under this Specific Aim, we will conduct a series of morphologic, anatomical, clinical, behavioral, and immunological analyses that will allow us to fully characterize this new Tg mouse model and to identify any pathologies or abnormalities. At the conclusion...