FIGURE 4.

EZ-TD inhibits HIV-1 vector release. (A) COS7 cells were transfected with the HIV-1 vector construction plasmids together with pcDNA3.1, EZ-Wt, EZ-TA, or EZ-TD expression plasmid, and culture supernatants from the transfected cells were inoculated onto HeLa/CD4 cells. Transduction titers of pcDNA3.1-transfected cells were always set to 1, and relative values of transduction titers of other transfected cells are indicated. Error bars show standard deviations, and asterisks indicate statistically significant differences compared to pcDNA3.1-transfected cells. (B) Cell lysates (left panels) and virion pellets of culture supernatants from the transfected cells (right panels) were analyzed by western immunoblotting using the anti-VSV-G epitope, anti-HIV-1 p24, or anti-actin antibody. Virion pellets were also separated on Phos-tag reagent-containing gels. (C) Cell lysates from Jurkat, 293T, HeLa, and COS7 cells were analyzed by western immunoblotting using the anti–phosphorylated-ezrin (upper panel) or anti-ezrin (lower panel) antibody. (D) Cell lysate from Jurkat cells transfected by the EZ-TD expression plasmid were applied to Phos-Tag reagent-containing gel, and then analyzed by western blotting using the anti-VSV-G epitope antibody.