Efforts to improve therapy for HIV have been hindered by a dearth of understanding of the mechanism by which HIV induces death of CD4+ T cells, the population it selectively depletes to cause AIDS. The majority of cells that die during HIV-1 infection are known as bystander T cells, since they are not productively infected. Therefore, understanding the mechanism by which HIV mediates T cell death in the bystander population is critical to identifying new potential therapeutic targets for preventing T cell death and immunodeficiency. Recent work by others suggests that pyroptosis, an inflammatory form of programmed cell death that culminates in cell lysis and release of inflammatory cytokines, may mediate bystander cell killing. A known trigger of pyroptosis is the efflux of potassium. Preliminary studies in our laboratory indicate that HIV infection increases expression of genes whose gene products mediate K+ efflux, and further that inhibition of K+ channels may decrease bystander cell pyroptosis in human lymphoid aggregate cultures (HLACs) and peripheral blood lymphocytes (PBLs). A candidate mediator of bystander killing is the HIV protein transactivator of transcription (Tat), since it is secreted by infected cells and can be readily taken up by bystander cells, and is also recognized to increase transcription of a number of cellular genes. Indeed, treatment of PBLs with Tat leads to massively increased expression of more than a dozen ion channels, including K+ channel genes KCNK17 and KCNE1; HIV infection also increases expression of these genes. Furthermore, preliminary evidence suggests that Tat has a functional impact on K+ channels by increasing K+ efflux in T cell lines. In view of the above, it is our hypothesis that Tat positively contributes to HIV-mediated bystander killing by pyroptosis by activating K+ channels. To test this hypothesis, we propose two aims. First, we will elucidate the role of K+ channels in HIV-1-mediated bystander T cell death. Our working hypothesis is that Tat/HIV-1-induced increases in ion channel expression increase susceptibility of bystander CD4+ T cells to death by pyroptosis. To test this working hypothesis, we will pharmacologically inhibit K+ channels and genetically knock down specific K+ channels using shRNA and assess whether cell death decreases in HIV infection of PBLs and HLACs by flow cytometric and Western blotting methods. In our second aim, we will define the mechanism by which Tat activates K+ channels. We hypothesize that Tat increases K+ efflux both by transcriptional induction of K+ channel genes and by transient activation of channels through signaling at the plasma membrane. We will employ luciferase reporter assays and mutational analysis to assess the transcriptional activity of Tat at channel promoters, and use patch clamping studies to assess the functional effect of Tat on K+ channel activity in PBLs and HLACs. Through these studies, we expect to identify specific ion channels as potential therapeutic targets for slowing or preventing immunodeficiency due to HIV infection.
A major barrier to developing new therapies against HIV has been a poor understanding of how HIV induces death of the cell populations it depletes to cause AIDS. The goal of this project is to investigate the hypothesis that the HIV Tat protein contributes to a form of inflammatory cell death known as pyroptosis that may be important for how HIV kills cells by activating potassium channels. This work could potentially identify potassium channels as targets for new antiretroviral therapies.
