fig9

Figure 9. HIV-1-infected h-microglia release Nef primarily via extracellular vesicles. (A) EVs and virions pelleted from 5-day-old h-microglia cultures infected with VSV-G pseudotyped YU-2, NL4-3, NL4-3Δnef, or left uninfected (-) were further separated by 6%-18% iodixanol density gradient ultracentrifugation, and eleven fractions were collected. To support efficient separation of EVs and virions on the gradient; (B) AChE activity (y-axis) of fractions (x-axis) was quantified by colorimetric assay and (C) p24 concentration (y-axis) in fractions (x-axis) was quantified by HIV-1 p24 ELISA assay; (D) Immunoblot analysis of precipitated fractions using antibodies against AChE, Nef, p24, and Flotillin. EV: Extracellular vesicles; AChE: acetylcholinesterase; Nef: negative factor; Flotillin: flotillin protein; HIV-1: human immunodeficiency virus type 1; h-microglia: human microglia; VSV-G: vesicular stomatitis virus glycoprotein; YU-2: HIV-1 strain YU-2; NL4-3: HIV-1 strain NL4-3; NL4-3Δnef: HIV-1 NL4-3 strain with Nef gene deletion; p24: HIV-1 core protein p24; ELISA: enzyme-linked immunosorbent assay.