Figure 7.

The specific silencing of TDP-43 in virus-producing cells decreases the protein level of HDAC6, stabilizing acetylated α-tubulin and HIV-1 Pr55^Gag and Vif viral proteins, thereby enhancing the production of viral particles and their infectivity. (A) The biochemical western blot analysis of TDP-43, HDAC6, acetylated α-tubulin, HIV-1 Pr55^Gag, and Vif proteins in virus-producing HEK-293T cells treated with a pull of siRNA oligonucleotides specific against TDP-43 (siRNATDP-43) or in the control (scrambled-treated cells). Total α-tubulin for protein load control is shown under each experimental condition. Data are represented as the mean ± S.E.M. of three independent experiments (see replicate data in Figure S7A, Supplementary Materials section). Right: Histograms show the quantification of western blot bands for the TDP-43/, HDAC6/, Pr55^Gag/, and Vif/α-tubulin ratios under this experimental condition. Data associated with the quantification of replicates are shown in Figure S7A, Supplementary Materials section. (B) Left: ELISA-HIV-1 p24 protein quantification of produced viral particles and siRNA-TDP-43- or scrambled-treated virus-producing HEK-293T cells. The ELISA-p24 technique was standardized with respect to the viral production control, which corresponded to the experimental condition of the scrambled-treated cells. Data are mean ± S.E.M. of three independent experiments. Right: HIV-1 infection was then carried out with synchronous doses of luciferase-pseudoviruses produced under the experimental conditions of panels (A,B) in the permissive CEM.NKR-CCR5 CD4+ T cells. CD4-treated cells showed the neutralization of HIV-1 infection by an anti-CD4 Ab. Data are mean ± S.E.M. of three independent experiments. (C) The biochemical western blot analysis of the content of Pr55^Gag, p24, and Vif proteins of the pseudoviruses used to infect permissive cells of the experiment of panel (B). A representative experiment of three is shown (see replicate data in Figure S7C, Supplementary Materials section). The quantification of the viral content in Pr55^Gag and Vif proteins, normalized by the p24 protein as a control for synchronous viral input, is shown in the right histograms, under each experimental condition. Data associated with the quantification of replicates are shown in Figure S7C, Supplementary Materials section. In (A–C), when indicated, * p < 0.05 and ** p < 0.01 are the p values for Student’s t test; a.u., arbitrary light units.