Figure 6: Depletion of eEF2 neutralizes Gag.
(a) Scheme of the experimental protocol. (b) HeLa cells were treated with siNS (left panel) or sieEF2 (middle panel) and transfected with pNL4-3 or treated with sieEF2 and co-transfected with pNL4-3 and eEF2-siRNA resistant (Flag-eEF2R; right panel). Cells were fixed and stained for G3BP1, eIF3b, Flag (eEF2) and Gag as indicated. Scale bar, 20 μm (c) Quantification of SG in cells from b. Data are presented as mean±s.d. from three separate experiments with at least 100 cells each analysed. Statistical analysis was performed with a one-way analysis of variance (ANOVA) combined with the Dunnet’s post test (**P<0.01 versus siRNA PatA and #P<0.01 versus siRNA Ars). (d) Cell lysates were analysed for Flag, eEF2, Gag and GAPDH. (e) Effects of depletion of eEF2 on intracellular Gag levels, virus production and infectivity. Graph shows relative change versus siNS-treated cells. Data are presented as mean±s.d. from three separate experiments. (f) HEK293T cells expressing HIV-1 in siNS, sieEF2 or rescue conditions were collected for polysome profile analysis and monitored by continuous ultraviolet absorbance at 254 nm. (g) RNA was isolated in each condition and subjected to RT-PCR. GAPDH mRNA was used as a control. (h) Intracellular vRNA was analysed using quantitative RT–qPCR. Transcript copy numbers per μg of intracellular RNA are shown. (i) Virion-associated RNA was isolated from cell culture supernatants of cells analysed in h. Transcript copy numbers per ml of cellular supernatant were obtained after RT–qPCR analyses. (j) The ratio of virion-associated and intracellular vRNA levels defines the encapsidation efficiency. Data are represented as mean±s.e.m. of three independent experiments. Statistical analysis was performed with a one-way ANOVA combined with the Dunnet’s post test (***P<0.001, **P<0.01, *P<0.05). (k) Left panel: spinning disk confocal microscopy of HIV-1-expressing HeLa cell. Cells were treated with sieEF2 and fixed. G3BP1, Gag and vRNA are identified by FISH/IF. Right panel: insets, 3D reconstruction with cutter edge of the region boxed. (l) Graph shows relative fluorescence intensities of G3BP1, Gag and vRNA within the cytoplasm or G3BP1 granules (n=10 cells). Data are represented as mean±s.d. Statistical analysis was performed with two-way ANOVA combined with the Bonferroni’s post test.
