ROLE OF EXOSOMES IN HIV-1 NEF RELEASE FROM GLIAL CELLS INFECTED WITH HIV-1 CONTRIBUTING TO DEVELOPMENT OF HIV/CNS DISORDERS

Abstract

HIV-associated CNS dysfunction is a significant problem among people with HIV (PWH), who now live longer due to viral suppression from combined anti-retroviral therapy (cART). Over the course of infection, HIV generates toxic viral proteins and induces inflammatory cytokines that have toxic effects on neurons in the CNS. Among these viral proteins, HIV Nef has been found in neurons of postmortem brain specimens from PWH. However, the source of Nef and its impact on neuronal cell homeostasis are still elusive. Using a simian immunodeficiency virus (SIV) infected rhesus macaque model of neuroHIV, we find SIV Nef reactivity in the frontal cortex, hippocampus and cerebellum of SIV-infected animals using immunohistochemistry (IHC). Interestingly, SIV-infected macaques treated with ART also showed frequent Nef positive cells in the cerebellum and hippocampus. Using dual quantitative RNAscope and IHC, we observed that some cells were positive for Nef, but were not for SIV RNA, suggesting that Nef protein was not limited to the cells that are actively infected with SIV. Using cell specific markers, we observed Nef protein in microglia/macrophages and astrocytes. Importantly, we also identified a number of NeuN-positive neurons, which are not permissive to SIV infection, but contained Nef protein. Further characterization of Nef-positive neurons showed caspase 3 activation, indicating late stage apoptosis in the CNS neurons. Moreover, we show that Nef is expressed in postmortem brain specimens from PWH. Interestingly, extracellular vesicles released from HIV infected astrocytes carry Nef. Further studies of the possible impact of these Nef-EVs on neurons in primary culture models revealed that Nef-carrying EVs were readily taken up by neurons, a significant amount of Nef was enriched in mitochondrial fractions, induced oxidative stress, increased ROS and dysregulated neuronal homeostasis. Overall, our study highlights the role of HIV secretory protein Nef in HIV CNS disease seen in PWH.