RNA helicase DDX3 regulates HIV-1 Rev function in astrocytes.

<p>(<b>A–B</b>) Astrocytes (HFA and SVGA) and HIV-1-permissive cells (Jurkat, HEK, and HeLa) were cultured in 6-well culture plates, and 24 h later, lysed and processed for Western blotting. Natural expression levels of RNA helicases (DDX1, DDX3, and RHA), HAX-1, Sam68, PKR, and TRBP in SVGA and HFA are shown for comparison with HIV-1-permissive cells. (<b>C</b>) SVGA were cultured in 6-well culture plates and, 24 h later, transfected with DDX3-HA expression vector or control vector. After 48 h, immunostaining for DDX3 was done in transiently transfected astrocytes using HA tag antibody (<b>inset</b>). In another set, SVGA-LTR-gagGFP cells were seeded in 12-well culture plates and transfected with Tat+Rev, Tat+Rev+antisense-DDX3 (AS), or pcDNA vector (control). Leptomycin-B was used a positive control for inhibition of Rev function. (<b>D</b>) Ectopic DDX3 expression partially reinstated AS-ablated Rev function. Cultured SVGA-LTR-gagGFP reporter cells in 12-well plates were transfected with Tat+Rev expression vector alone or in combination with DDX3, AS, or mutant DDX3 (d-DDX3, functionally dead mutant). In parallel, reporter cells were transfected with control empty vector. Leptomycin B was used as a positive control for inhibition of Rev function. At 30 h after transfection, GFP-positive cells were counted in 10 random fields in duplicate sets and plotted. (<b>E</b>) HeLa and SVGA cells in duplicate were transfected with HIV-1 NL4-3 infectious molecular clone (pNL4-3) alone or in combination with AS or d-DDX3 expression vectors. In parallel, cells were transfected with empty vector (control). At 48 h after transfection, p24 in supernatants was monitored in triplicate by ELISA (p<0.004 and p<0.009) (n = 2).