Figure 6

Vimentin filament collapse relieves M2BP inhibition of HIV-1 virion production.

(A–D) HEK293T cells were transfected with the HIV-1 vector producing plasmids indicated, together with plasmids expressing M2BP and VIM_mut. At 12 h posttransfection, chemicals indicated were added to the culture media. At 36 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells or analyzed for protein expressions. At 48 h postinfection, luciferase activity was measured. (A,C) The relative luciferase activity in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. (B,D) Protein expressions in the cell lysates and culture supernatants. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are representative of three independent experiments. (E) An empty vector or a plasmid expressing VIM_mut was nucleofected into MT4-M2BPi cells together with the HIV-1-producing plasmids indicated. A plasmid expressing firefly luciferase was included to serve as a control for transfection efficiency and sample handling. At 8 h posttransfection, IFNα-2b was added to the culture media. At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes of the culture supernatants were used to infect HEK293T cells. At 48 h postinfection, luciferase activity was measured in the recipient cells (lower panel). Relative CA levels and relative luc activity in the MT4-Ctrli cells without IFN treatment was set as 100. Data presented are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, n.s. p > 0.05