Slp2a is a Rab27a effector required for PM delivery of PI4KIIα and HIV-1 assembly. (A) p24 production by Jurkat cells transduced with different shRNAs targeting Rab27 effector genes and subsequently infected with a VSV-G–pseudotyped HIV-1 strain was evaluated at day 4 p.i. Data are the means ± SD of three independent experiments. Solid lines and dashed lines indicate 100% and 50%, respectively, of p24 production by control cells. (B) Inhibition of gene expression of EXPH5 and SYTL3 was determined by qPCR (n = 2). (C) Inhibition of Slp2a expression was determined by immunoblotting. (D) Kinetics of HIV-1 (strain IIIB; inoculum: 100 ng/ml) production by Jurkat cells stably transduced with control shRNA (closed circles), SYTL2 shRNA 1 (open squares), EXPH5 shRNA1 (open circles), and SYTL3 shRNA 1 (open triangles). Rab27a-silenced cells were included for comparative purposes (closed squares). One representative experiment of two performed in triplicates is shown. (E) Intracellular distribution of Gag in control or Slac2-b–, Slp2a-, and Slp3-silenced cells was analyzed by LSCM at day 7 p.i. (F) LSCM visualization of cells transiently transfected with dsRed-Rab27a and Slp2a-GFP. (G–J) LSCM of live control and Rab27a-silenced cells stably expressing CD63-GFP (G), PI4KIIa-GFP (H), GFP-P4M (I), and PH-GFP (J). Representative confocal images (left) and transmitted light images (right) are shown. Quantifications were performed by blinded observers on a per-cell basis, in ≥50 cells of each condition. (K) LSCM of live Slp2a-silenced Jurkat cells transiently transfected with PI4KIIα-GFP and mCherry-CD63 plasmids. Error bars show SDs. KD, knockdown. Bars, 2 µm.