Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Despite suppressive combination anti-retroviral therapy (cART), HIV infection persists in tissue reservoirs. For example, in the central nervous system (CNS), expression of viral transcripts has been observed in cART- suppressed patients, which likely contributes to chronic immune activation, the chief driver of HIV-associated neurocognitive disorders (HAND). Persistent inflammation is presumed to cause immune dysfunction and degradation of immune control mechanisms, likely contributing to virus persistence. Thus, there seems to be a positive feedback loop of inflammation and viral persistence. To reduce the size of viral reservoir and achieve a functional HIV cure, therefore, it is crucial to understand mechanisms that contributes to chronic inflammation. Microglia, the CNS-resident microphages, are thought to be a major cellular reservoir of HIV-1 because of their self-renewal capacity. We recently demonstrated that cytoplasmic expression of HIV intron-containing RNAs (icRNAs) induced IFN-I-dependent pro-inflammatory responses in iPSC (induced pluripotent stem cell)-derived human microglia. We therefore hypothesize that de novo HIV icRNA expression in HIV-infected microglia drives persistent inflammation and immune dysfunction, leading to degradation of immune control mechanisms and contributing to virus persistence. To date, however, how HIV infection triggers microglia activation and if microglia activation affects viral persistence remain unclear. In this proposal, we will investigate mechanisms of HIV-icRNA-induced innate immune activation in microglia and their contribution to immune dysfunction with the following aims. In Aim1, we will identify the host machinery sensing HIV icRNA in microglia using two complementary mass spectrometry (MS)-based approaches in collaboration with Dr. Sherer at University of Wisconsin-Madison. We will purify HIV-icRNA-associated proteins by hybridization purification. In addition, we will use biotin ligase-mediated proximity labeling strategy to identify host proteins in close contact with HIV-1 icRNA. We will then perform MS analysis to identify the sensor. We will validate the role of these candidate proteins in microglia activation using human iPSC-derived microglia. In Aim2, we will investigate the role of soluble factors produced from HIV-infected microglia in immune dysfunction. We will test if HIV infection in iPSC-microglia leads to production of soluble factors such as pro-inflammatory cytokines, complement 4A, and peptidyl arginine deiminase 2. We will then knock out candidate genes by CRISPR/Cas9 in iPSCs, followed by differentiation to microglia, and test if HIV infection in KO microglia leads to dysfunction. We hope that successful completion of this proposal will not only help deepen our knowledge on HIV RNA-host interaction, but also contribute to future development of novel therapeutic modalities that reduce HIV RNA- induced immune activation and virus persistence in the CNS.
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