Fig 7. MyoF restricts lysosomal degradation of Env.

(A) Levels of endosomal proteins in HTLV-1 infected cells following WJ460 treatment. SLB-1 cells were treated with DMSO or 1 μM WJ460 for 24 h. Whole cell extracts (25 to 50 μg) were analyzed by Western blot using antibodies against the indicated proteins. (B) Knockdown of MyoF expression reduces the level of caveolin-1 (Cav1). Whole cell extracts (25 to 50 μg) were prepared from SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA and analyzed by Western blot using antibodies against the indicated proteins. (C) Knockdown of MyoF expression does not affect the level of SU (gp46) at the cell-surface. SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA were labeled with a primary antibody against SU/Pr followed by an APC-conjugated secondary antibody, fixed and analyzed by flow cytometry. Histograms are representative of two independent experiments and show relative cell surface labeling as follows: secondary antibody alone (CT) and SU-labeled shGFP cells, light gray and light blue, respectively; secondary antibody alone (CT) and SU-labeled shMYOF cells, dark gray and dark blue, respectively. (D) Inhibition of lysosomal proteases partially restores intracellular levels of SU (gp46) in HTLV-1 infected cells treated with WJ460. SLB-1 cells were treated with DMSO or 1 μM WJ460 with or without 5 mg/mL leupeptin (L) and 25 μM E64D (E) for 24 h. Whole cell extracts (50 μg) were analyzed by Western blot using antibodies against β-actin and SU/Pr. (E) Knockdown of MyoF expression increases SU (gp46) association with lysosomes. SLB-1 cells stably expressing an shRNA targeting GFP (negative control) or MYOF mRNA were fixed, permeabilized, labelled with Alexa Fluor 488- and Alexa Fluor 594-conjugated antibodies against SU/Pr and LAMP-2, respectively, and analyzed by confocal microscopy. The images show 3D projections constructed from z-stacks comprised of 0.57 μm optical slices. The graph shows Manders’ Colocalization Coefficients (MCC) for the fraction of SU overlapping LAMP-2; ** p<0.01. (F) Inhibition of endosomal trafficking reduces HTLV-1 infection. SLB-1 cells were treated with DMSO, 10 μM EGA, 10 μM vacuolin-1 (Vac-1) or 1 μM apilimod (Api) for 4 h prior to co-culture with CHOK1-Luc cells. The graph shows luciferase assay results averaged from three replicates for each condition of a single experiment and are representative of three independent experiments; ** p<0.01.