Figure 8.

Depletion of 90K in primary HIV-1 target cells enhances particle infectivity, enhances Env content of HIV-1 particles, and accelerates HIV-1 spread. (A) VSV-G/HIV-1 _NL4.3-infected macrophages were transfected one day post-infection with a 90K-specific or an irrelevant siRNA. At day 7 post infection, particle infectivity of released HIV-1 was calculated as the ratio of infectivity to p24 capsid antigen. Cell lysates and sucrose cushion-purified virus were analyzed by Western Blotting. Percentages indicate the relative gp120 incorporation and the efficiency of gp120 processing, calculated as in Figure 5(G-H) (B) Macrophages obtained from two donors were siRNA-transfected and subsequently infected with HIV-1_Ba-L. One day post infection, excess virus was removed by thorough washing. Supernatants were subjected to anti-p24 capsid antigen ELISA at indicated time points. Aliquots of cells were taken for 90K knockdown validation over time by Western Blotting. Shown is 90K expression at day 13 post infection. (C) Macrophages were infected with HIV-1_Ba-L and simultaneously treated with IFN-α (100 U/ml). Supernatants were harvested at the indicated time points, frozen, and analyzed for released p24 capsid and (D) infectious HIV-1. Shown are arithmetic means of triplicates ± S.D. (E) Using the data from (C) and (D), the particle infectivity was calculated. Data points indicate arithmetic means of triplicates ± S.D. (F) Cell lysates were subjected to Western Blotting using indicated antibodies. (G) IL-2/PHA-stimulated PBMCs from two donors were nucleofected with a 90K-specific or an irrelevant siRNA followed by HIV-1_NL4.3 infection over night. Excess virus was removed by thorough washing at indicated time points supernatants were subjected to p24 capsid antigen ELISA. 90K knockdown at the time point of infection was validated by 90K ELISA. * : p < 0.05; **: p < 0.02 (Student’s T-Test).