Figure 10. TRIM28 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.

(A) shRNAs targeting luciferase and TRIM28 were packaged into lentiviruses and infected CD4⁺ T cells from HIV-1-infected individuals. Unstimulated CD4 +T cells were used as negative control (NC). Stimulation with αCD3/αCD28/IL-2 was used as positive control. Intracellular HIV-1 RNA was isolated and quantitated by qPCR. Experiments were conducted in three HIV-1-infected individuals. (B) The experiment setting was as in (A). Envelope V1 to V3 region from intracellular HIV-1 RNAs was reverse-transcribed and PCR-amplified. The PCR products were TA-ligated in pMD-18 T vector. At least 60 single clones were picked from each group and sequenced. The sequences from each group were aligned and the genetic diversity index was calculated and analyzed by Mann-Whitney U-test. The upper panel showed the statistical analysis results. The lower panel indicated the bootstrap consensus trees which were generated based on HIV-1 sequences. *p<0.05, **p<0.01, ***p<0.001. (C) Resting CD4⁺ T cells from HIV-1-infected individuals were isolated and nucleofected with siRNAs targeting negative control or TRIM28. Seventy-two hours later, PHA-stimulated uninfected CD4⁺ T cells were added into each group and co-cultured for another 27 days. The supernatants were collected and half-changed every 3 days. P24 antigens in supernatants were measured with ELISA and plotted in log₁₀ scale. Dashed lines indicated the limit of detection (L.O.D.) of 50 pg/ml. Triplicates were represented by mean ±SEM. (D) Schematic of TRIM28-mediated HIV-1 latency.

Figure 10—figure supplement 1. Cytotoxicity assay, cell viability assay and cell number counting used to evaluate the toxicity of targeting TRIM28.

(A) TRIM28 in Hela cells and HIV-1-infected CD4⁺ T cells was knocked down by siRNA targeting TRIM28. ShRNA and sgRNA lentiviruses targeting TRIM28 were used to knock down TRIM28 and knock out TRIM28 in J-Lat 10.6 respectively. Cell Counting Kit-8 (CCK-8) reagents were incubated with wild type and TRIM28-deficient cells for 3 hr followed by measuring the absorbance at 450 nm using a microplate reader. Fold changes of absorbance in each group were normalized to wild-type groups. (B) The experiment setup was conducted as in (A). The percentages of viable cells were quantitated every 2 days by measuring the percentages of amine-reactive fluorescent dye non-permeant cells. (C) The experiment setup was conducted as in (A). Cell numbers were recorded every 2 days for both wild-type and TRIM28-deficient cells.

Figure 10—figure supplement 2. Cell proliferation assay used to evaluate the toxicity of targeting TRIM28.

(A–D) The experiment setup was conducted as in Figure 10—figure supplement 1. On Day 0, cells from each group were stained with CFSE. The percentage and mean fluorescence intensity (MFI) of CFSE-positive cells were analyzed by flow cytometry every 2 days.

Figure 10—figure supplement 3. TRIM28 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.

(A) Schematic of experiments on primary CD4 +T cells from HIV-1-infected individuals. (B) The knockdown efficiency of shTRIM28 in HIV-1-infected CD4⁺ T cells. (C) The knockdown efficiency of nucleofection of siRNAs targeting TRIM28. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05, **p<0.01.

Figure 10—figure supplement 4. SUMO4 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.

(A) SiRNAs targeting NC and SUMO4 were nucleofected into CD4⁺ T cells from HIV-1-infected individuals. Unstimulated CD4⁺ T cells were used as Mock. Stimulation with αCD3/αCD28/IL-2 was used as positive control. Intracellular HIV-1 RNA was isolated and quantitated by qPCR. Experiments were conducted in three HIV-1-infected individuals. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. (B) The knockdown efficiency of nucleofection of siRNAs targeting SUMO4. Data represents mean ±SEM in triplicates. p-Values were calculated by Student’s t-test. *p<0.05.