Resistance to Apoptosis in HIV-Infected astrocyte as a potential mechanism for viral Persistence in CNS

DIEGO OJEDA; JORGE QUARLERI; MAURICIO CAROBENE

Washington

Simposio; The 12th International Symposium on NeuroVirology; 2013

International Society for NeuroVirology

BackgroundApoptosis plays a critical role in HIV-1-associated neuropathogenesis. HIV-1 has developed multiple strategies to modulate apoptosis related-pathways on infected cells thus promoting its survival and favoring its role as viral reservoirs. HIV-1 establishes infection in astrocytes of the CNS causing minimal cytopathology in these long-lived cells. This study aimed to evaluate the HIV infection impact on apoptosis cascade in infected astrocytes and their neighboring uninfected cells. MethodsAn HIV infectious molecular clone pNL43-derived and tagged with the GFP (named HIV-GFP) was constructed. It was also pseudotyped with the VSV-G glycoprotein by co-transfection of 293T cells.U373 human astrocytic cells were used as in vitro models for astrocyte infection. To detect apoptotic events at 1 to 6 days post infection (dpi), co-staining with 7AAD and annexin-V was performed by triplicate. Single-cell analysis aimed at simultaneously identifying apoptotic-infected (Ann+/GFP+) and apoptotic-uninfected (Ann+/GFP-) neighbor cells by FACS. The values were expressed as mean±SD. Statistic analysis was performed by Student T test and p values 0.005) between productively infected astrocytes (13±7.7 and 7.3±9.7 GFP+, respectively) and neighbor uninfected cells (16.5±9.5 and 7.65±6.5 GFP-, respectively). In contrast, when comparing the level of apoptosis (annexin-V+) at 3, 4, 5 and 6 dpi between infected vs. neighbor uninfected astrocytes (GFP+/GFP-) a significant decreased (p