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Figure 8 | Retrovirology

Figure 8

From: Ubiquitin conjugation to Gag is essential for ESCRT-mediated HIV-1 budding

Figure 8

Incorporation of Gag-Ub into assembly sites alleviates DUb-ESCRT inhibitory effects. A) 293T cells expressing EIAV (lane 1),were also transfected with increasing amounts of EIAV Gag-Ub expression vector (500 ng and 1μg) (lanes 2 and 3), with Flag-DUb-Alix alone (100 ng) (lane 4), in combination with increasingamounts of Gag-Ub (500 ng and 1μg) (lanes 5 and 6), or with inactive DUb*-Alix alone (100 ng) (lane 7) whereas lane 8 shows expression of Gag-Ub alone. Virions and cells were harvested 24 h post-transfection and their protein contents analyzed by WB using an anti-EIAV antibody. Alix and DUb/DUb*-Alix fusion proteins were detected by an anti-Alix antibody. B) 293T cells expressing HIV-1 YP- (lane 1), were also transfected with increasing amounts of HIV Gag-Ub expression vector (1 and 2 μg) (lanes 2 and 3), with DUb-TSG101 alone (200 ng) (lane 4), or in combination with increasing amounts of HIV Gag-Ub expression vector(1 and 2 μg) (lanes 5 and 6) whereas lane 7 shows expression of HIV Gag-Ub alone (1 μg). Virions and cells were harvested 24 h post-transfection and their protein contents analyzed by WB using an anti-p24CA antibody. TSG101 and DUb-TSG101 fusion protein were detected with an anti-TSG101 antibody.

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