Fig. 6: CHMP2B K6 and its methylation contribute to HIV particles release.

HeLa cells expressing GFP, CHMP2B_WT-GFP, CHMP2B K6R-GFP and CHMP2B K6A-GFP were transfected with pNL4-3 HIV-1 pro-viral DNA. Forty-eight hours later, cells were lysed and virus contained in supernatants were pelleted by ultracentrifugation. Viral production was evaluated by western blot and by ELISA quantification of HIV-1 CAp24. A Western blot analysis of HIV-1 Gag and CAp24 products, GFP and GAPDH in transfected cells and pelleted viruses. B HIV-1 release index was calculated as the ratio between released CAp24 and cell-associated CAp24. Results were normalized to control cells (set as 100%). C The virus titer was scored by infection of HeLa P4R6 indicator cells, followed by β-galactosidase activity quantification in the cells. (Statistical analysis using two-tailed unpaired Student’s t test, ns non-significant (p > 0.05), N = 4, mean ± SD. All western blots are representative of at least three independent experiments). D HeLa cells transfected with indicated siRNA were transfected with pNL4-3 HIV-1 pro-viral DNA. Forty-eight hours later, cells were lysed, and virus contained in supernatants were pelleted by ultracentrifugation. Viral production was evaluated by western blot and by ELISA quantification of HIV-1 CAp24. Western blot analysis of HIV-1 Gag and CAp24 products, SMYD2 and GAPDH in transfected cells and purified viruses. E HIV-1 release index was calculated as the ratio between released CAp24 and cell-associated CAp24. Results were normalized to control cells (set as 100%). F The virus titer was scored by infection of HeLa P4R6 indicator cells, followed by β-galactosidase activity (Statistical analysis using two-tailed unpaired Student’s t test, N = 4, mean ± SD. All western blots are representative of at least three independent experiments).