Figure 3. USP49 enhances the expression of A3G even in absence of Vif.

(A) HEK293T cells were transfected with A3G-GFP-expressing plasmid and siRNA specific for USP49. MG132 was treated for 12 hr before harvest. The GFP expression was detected with a PE Envision at 48 hr post-transfection. Error bars represent the SEM of three independent experiments. **p<0.01. (B) HEK293T cells were transfected with A3G-HA-expressing plasmid and siRNA specific for USP49. MG132 was treated for 12 hr before harvest. After 48 hr post-transfection, cells were lysed and Western blot was performed with the indicated antibodies. Representative data were shown and plotted with at least three independent experiments.* p<0.05. (C) HEK293T cells were transfected with A3G-HA-expressing plasmid, and different amounts of USP49-Flag-expressing plasmid. After 48 hr, cells were lysed and Western blot was performed with the indicated antibodies. Representative data were shown and plotted with at least three independent experiments. (D) HEK293T cells were transfected with pcDNA3.1-A3G-HA, pcDNA3.1-USP49-Flag, and pNL4-3ΔVif. After 48 hr, cell pellets and supernatants were collected respectively. Cell pellets were lysed and subjected to immunoblotting with anti-HA, anti- Flag, and anti-GAPDH antibodies. Viral particles were collected from filtered supernatants by ultracentrifugation. The pelleted viral particles were lysed and detected by western blotting with anti-HA and anti-p24 antibodies. (E) HEK293T cells were transfected with pcDNA3.1-A3G-HA, pNL4-3, and a USP49-specific siRNA, After 48 hr, cell pellets and supernatants were collected respectively. Cell pellets were lysed and subjected to immunoblotting with anti-HA, anti- Flag, and anti-GAPDH antibodies. Viral particles were collected from filtered supernatants by ultracentrifugation. The pelleted viral particles were lysed and detected by western blotting with anti-HA and anti-p24 antibodies. (F) HEK293T cells were transfected with pcDNA3.1-A3G-HA and a USP49-specific siRNA. MG132 (10 uM) or MLN4924 (20 uM) was treated for 12 hr before harvest. After 48 hr post-transfection, cells were lysed and Western blot was performed with the indicated antibodies. Representative data were shown and plotted with at least three independent experiments.* p<0.05, **p<0.01. (G) HEK293T cells were transfected with pcDNA3.1-A3G-GFP, pcDNA3.1-USP49-FLAG and indicated siRNAs. The GFP expression was detected with a PE Envision at 48 hr post-transfection. Error bars represent the SEM of three independent experiments.

Figure 3—figure supplement 1. A3G can be packaged into HIV-1 virions with the help of USP49.

HEK293T cells were transfected with pcDNA3.1-A3G-HA, pcDNA3.1-USP49-Flag, and pNL4-3. After 48 hr, cell pellets and supernatants were collected respectively. Cell pellets were lysed and subjected to immunoblotting with anti-HA, anti- Flag, and anti-GAPDH antibodies. Viral particles were collected from filtered supernatants by ultracentrifugation. The pelleted viral particles were lysed and detected by western blotting with anti-HA and anti-p24 antibodies.

Figure 3—figure supplement 2. Detection of the knockdown efficiency of various siRNAs.

(A) HEK293T cells were transfected with indicated siRNAs. After 48 hr, the total RNA was extracted with TRIZOL and the mRNA expression of each gene was detected by qRT-PCR. Error bars represent the SEM of three independent experiments. (B) HEK293T cells were transfected with pcDNA3.1-A3G-GFP, pcDNA3.1-Vif, and indicated siRNAs. The GFP expression was detected with a PE Envision at 48 hr post-transfection. Error bars represent the SEM of three independent experiments. (C) HEK293T cells were transfected with pcDNA3.1-AID-GFP and the indicated siRNAs. The GFP expression was detected with a PE Envision at 48 hr post-transfection. Error bars represent the SEM of three independent experiments.