Figure 5. Mutations within hVps4 affect HIV-1 budding and ESCRT-III disassembly in vitro.

(a) 293T cells were cotransfected with HIV-1 proviral DNA and vectors expressing GFP fused to wild-type (WT) hVps4A or to the indicated hVPS4A mutants. Virus release (upper left panel) and intracellular Gag processing were examined by western blotting with an anti- HIV-1 CA antibody (upper right panel). The expression levels of WT and mutant GFP–VPS4A were compared by western blotting with an anti-GFP antibody (lower panel). (b) Disassembly of fluorescein-labelled ESCRT-III CHMP2AΔC-CHMP3 helical tubes measured by change in fluorescence emission intensity on addition of VPS4B plus ATP and Mg²⁺ (red); MsVps4 plus AMP-PNP and Mg²⁺ (red dashed line) and mutants of hVPS4B plus ATP and Mg²⁺ as indicated (I124E, green; M350A magenta dashed line; F160, magenta; R253A, yellow). The experiment was repeated three times.