289 HIV-1 Enters Primary Human Fetal Brain Microvascular Endothelial Cells by a Lipid Raft-independent Mechanism E. G. Argyris*, E. Acheampong, M. Mukhtar, K. J. Williams, R. J. Pomerantz Thomas Jefferson Univ, Philadelphia, PA
Background: Recent studies have demonstrated the significance of membrane cholesterol and lipid rafts in HIV-1 entry into permissive cells, including primary human brain microvascular endothelial cells (BMVECs) from adults. In this study, we examined the role of cellular cholesterol and lipid rafts in HIV-1 entry into primary human fetal BMVECs, which represent an important HIV-1 CNS-based cell reservoir and site of neuro-invasion.
Methods: Human fetal BMVECs were maintained and passaged in vitro under strict conditions established in our laboratory. JURKAT cells served as a positive control. The purity of BMVECs was analyzed by immunofluorescent staining with antibody against von Willebrand factor. Depletion of cellular cholesterol and dispersal of membrane lipid rafts was achieved by treatment of the cells with 10 mM of different cyclodextrins for 30’ at 378C in serum free medium. The cyclodextrin effect on the cells was analyzed by immunofluorescent staining with cholera toxin B-FITC. HIV-1 (NL4-3, YU2 and 89.6) binding, viral entry, and replication was analyzed by free virus binding assays at 48C and 378C, respectively. Cell lysates and supernatants were collected and HIV-1 p24 antigen was quantitated by ELISA.
Results: Our data from the free HIV-1 binding assays at 48C, as well as from the infectivity assays at 378C in BMVECs treated with cyclodextrins, demonstrate that membrane cholesterol and lipid rafts are not implicated in HIV-1 binding and viral entry into primary human fetal BMVECs. No differences were observed in binding and entry of various HIV-1 strains between BMVECs treated with cyclodextrins and cyclodextrin-untreated BMVECs. Our data also confirmed the important role of lipid rafts and cellular cholesterol in HIV-1 entry into JURKAT cells, which served as a positive control.
Conclusions: Our findings strongly suggest a cholesterol and lipid raft-independent mechanism in HIV-1 entry into primary human fetal BMVECs, which represent an important cellular reservoir in HIV-1 entry and infection of the CNS. The molecular mechanisms of viral entry and infection of CNS and its cellular components remains unclear. Further work is needed in order to fully characterize the pathways of HIV-1 entry into the CNS, involving not only BMVECs but also other critical CNS cellular components such as neurons, microglia, and astrocytes.
