Fig. 8
From: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells
HIV-1 Gag inhibits Tat palmitoylation by exporting Cyclophilin A. a PC12 cells were cotransfected with Tat-Flag and Gag (1/1 ratio) or an empty vector as indicated. Cells were then labeled overnight with 17-ODYA before assaying Tat palmitoylation using click chemistry. b Human primary CD4+ T-cells were infected with HIV-1 (NL4.3). After 24 h cells were processed for immunofluorescence detection of Tat and Gag, using fluorescent WGA to localize the plasma membrane and the TGN. When indicated, cells were pretreated with 5 mM neomycin for 1 h before fixation. Bar, 5 µm. c PC12 cells were cotransfected (1/1) with Tat-FLAG and a Gag mutant or an empty vector as indicated before assaying Tat palmitoylation using ABE. Gag-MA-22/27A and Gag-CA-G89V are mutated within their PI(4,5)P2- and CypA-binding motif, respectively. d Jurkat T-cells were transfected with the indicated Gag mutant using nucleofection. Transfection efficiency was ~80%. After 24 h, cells were lysed for Western blots. Triplicate blots were used for the quantification. Data are mean ± SEM (n = 3) and were analyzed using one-way ANOVA (***p < 0.001)
