Figure 7
Perturbation of actin polymerization enhances HIV-1 release from IPMC. (A) MDMs (4×105) were infected with HIV-1BaL for 7 days. Cells were washed in phosphate-buffered saline, new medium was added and virus released into the culture supernatant was collected at the indicated times, concentrated by centrifugation through a sucrose cushion and subjected to western blotting for HIV-1 Gag proteins. (B) MDMs were infected with HIV-1BaL for 7 days, washed and treated with DMSO (control) or 2 μM latrunculin A for 2 hours. Right hand panels: HEK293T cells were transfected with the HIV-1 molecular clone NL4.3-R3A, incubated for 48 hours and treated with DMSO (control) or 2 μM latrunculin A for 2 hours. Viruses released during treatment were concentrated by centrifugation and viral proteins detected by western blotting. The blots were imaged with a LAS4000 CCD camera and quantified using ImageQuant Software, as described in the Methods. Protein band intensities of treated samples were normalized to the DMSO control. The western blots are representative examples of five independent experiments, while the graphs show the average of five independent experiments. (C) MDMs infected for 7 days were treated simultaneously with the HIV-1 protease inhibitor amprenavir (PRI) and latrunculin A for 2 hours. The plot on the right shows band intensities of viral proteins quantified using ImageQuant software. The western blot is representative of four independent experiments and the plot shows the average of four independent experiments. Error bars represent standard deviations. Significance is shown as the difference between latrunculin-treated and control cells *P <0.05; **P <0.01.