Figure 10
From: The HDAC6/APOBEC3G complex regulates HIV-1 infectiveness by inducing Vif autophagic degradation
HDAC6-mediated proviral Vif degradation stabilizes A3G and impairs HIV-1 infectiveness. a Western blot of A3G degradation by proviral Vif in HEK 293T cells co-transduced with A3G-3xHA and HIV-1NL4.3 (dose–response) or HIV-1 Δvif provirus. b Effect of HDAC6 on HIV-1 infectiveness assayed in HeLa P5 cells incubated with equivalent luciferase-based X4-tropic pNL4-3.Luc.R-E- viral inputs produced in control, or in HEK 293T cells overexpressing A3G-3xHA, or overexpressing HA-wt-HDAC6 or HA-HDAC6-ΔBUZ together with A3G-3xHA. The control of infection (+) is represented by virus produced in HEK 293T, only expressing A3G-3xHA. A3G-3xHA and Vif proteins incorporated into nascent virions (p24) are shown. c Effect of HDAC6 on HIV-1 infectiveness, assayed in HeLa P5 cells incubated with equivalent R5-tropic pNL4-3.Luc.R-E- viral inputs [same experimental conditions as in (a)], and measured by β-galactosidase activity. d Effect of specific HDAC6 siRNA on HIV-1 infectiveness assayed in HeLa P5 cells incubated with equivalent R5-tropic viral inputs produced in siRNA-HDAC6-treated HEK 293T cells, and compared to control (+), scrambled-treated HEK 293T cells. HDAC6/α-tubulin and proviral-Vif/α-tubulin expression ratios are shown for this representative experiment. Vif/α-tubulin ratios from three independent experiments are shown in the right histogram. e Western blot of HDAC6-mediated proviral Vif degradation concomitant with p62, in control HEK 293T cells transduced or not with a pNL4.3-Luc-R-E- provirus, together with HA-wt-HDAC6, in the absence of A3G. 3-MA or MG132 treatment is shown under the same conditions. Tubulin-deacetylase activity of HDAC6, and proviral Pr55Gag and Vif proteins are shown. All western blots are from a representative experiment of three, and show input expression for endogenous HDAC6 (in control, scrambled- or siRNA-HDAC6-treated cells), overexpressed HA-wt-HDAC6 or HA-HDAC6-ΔBUZ, or A3G-3xHA and proviral Pr55Gag, p24 and Vif proteins. Infection assay were done in triplicate and results are representative of four independent experiments (mean ± S.E.M.; n = 12). When indicated, α-tubulin or actin is the control for total protein.