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. 2019 Aug 9;8:e48318. doi: 10.7554/eLife.48318

Figure 2. USP49 enhances the inhibitory effect of A3G on the infectivity of HIV-1.

(A) HEK293T cells were first transfected with USP49-specific siRNA. After 12 hr, cells were co-transfected with pcDNA3.1-A3G-HA, pcDNA3.1-Vif-HA, and pNL4-3ΔVif. Culture supernatants were harvested at 72 hr post-transfection and then infected TZM-bl cells. After 48 hr, cells were harvested and the HIV-1 infectivity was detected by luciferase assay. Error bars represent the SEM of three independent experiments. *p<0.05. (B) HEK293T cells were co-transfected with pcDNA3.1-A3G-HA, pNL4-3ΔVif, and plasmids expressing Vif-HA or USP49-Flag respectively. Culture supernatants were harvested at 72 hr post-transfection and then allowed to infect TZM-bl cells. After 48 hr, cells were harvested and the HIV-1 infectivity was detected by luciferase assay. Error bars represent the SEM of three independent experiments. *p<0.05, **p<0.01. (C) HEK293T cells were co-transfected with pcDNA3.1-A3G-HA, pNL4-3ΔEnv, and different amounts of USP49-Flag-expressing plasmid respectively. Culture supernatants were harvested at 72 hr post-transfection and then infected TZM-bl cells. After 48 hr, cells were harvested and the HIV-1 infectivity was detected by luciferase assay. Error bars represent the SEM of three independent experiments. **p<0.01, ***p<0.001. (D) HEK293T cells were transfected with pNL4-3ΔVif, pcDNA3.1-A3G-HA plus pNL4-3ΔVif, or pNL4-3ΔVif plus USP49-Flag plasmids respectively. Culture supernatants were harvested at 72 hr post-transfection and then allowed to infect TZM-bl cells. After 48 hr, cells were harvested and the HIV-1 infectivity was detected by luciferase assay. Error bars represent the SEM of three independent experiments. ***p<0.001. (E) The primary CD4+T cells were stimulated with anti-CD3/28 for 2 days and then nucleofected with indicated siRNAs. After 24 hr, cells were infected with pNL4-3ΔEnvΔVif-GFP pseudotyped viruses. The infectivity were detected by flow cytometr on 48 h.p.i. Representative data were shown and plotted with at least three independent experiments.* p<0.05, **p<0.01. (F) HEK293T cells were transfected with pNL4-3 or Vif-Y40H-mutated-pNL4-3 respectively. Culture supernatants were harvested at 72 hr post-transfection and then allowed to infect shRNA-KD-USP49 primary CD4+ T cells. Cell supernatants were harvested for detecting HIV-1 P24 by ELISA Kit on several time points. Error bars represent the SEM of three independent experiments. The difference of p24 production from HIV-1NL4-3VifY40H infection between shRNA-NC and shRNA-KD-USP49 in primary CD4+ T cells at several time points was statistically analyzed. **p<0.01, ***p<0.001.

Figure 2.

Figure 2—figure supplement 1. Analysis of A3G-induced hypermutation of prot DNA region.

Figure 2—figure supplement 1.

(A) G-to-A mutation loads in proviral DNA from viruses originally produced in Figure 2B (mean + /- SD of 3 independent experiments with a minimum of 10 sequences analyzed per condition). (B) G-to-A mutation loads in proviral DNA from viruses originally produced in Figure 2F (mean + /- SD of 3 independent experiments with a minimum of 10 sequences analyzed per condition).
Figure 2—figure supplement 2. The A3G protein level was detected by flow cytometry.

Figure 2—figure supplement 2.

The cells from Figrue 2E were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS/10% normal goat serum/0.3M glycine to block non-specific protein-protein interactions followed by the A3G antibody (Abcam, cat#ab75560) for 30 min at 22°C. The secondary antibody used was DyLight 594 goat anti-mouse IgG (H+L) at 1/500 dilution for 30 min at 22°C. Then these cells were detected by flow cytometry and acquisition of >5000 events was performed. Representative data were shown (A) and error bars represent the SEM of three independent experiments (B).