Figure 4. H₂S donor (GYY4137) suppresses HIV-1 reactivation and replication.

(A) U1 cells were treated with NaHS or GYY4137 and media supernatant was harvested to assess H₂S production by methylene blue assay over time. (B, C) U1 cells were pre-treated with 5 mM GYY4137 or 5 mM NAC for 24 hr and then stimulated with 5 ng/ml PMA for 24 and 48 hr. Total RNA was isolated and HIV-1 reactivation was assessed by gag RT-qPCR (B). Culture supernatant was harvested to monitor HIV-1 release by p24 ELISA (C). (D) U1 cells were pretreated with 5 mM GYY417, 5 mM NAC for 24 hr or left untreated and then stimulated with 100 ng/ml TNF-ɑ for 24 and 48 hr. HIV-1 reactivation was assessed by gag RT-qPCR. (E) U1-shCTH and U1-shNT were pretreated with 5 mM GYY4137 for 24 hr and stimulated with 5 ng/ml PMA for 24 hr. Culture supernatant was harvested to determine HIV-1 reactivation by HIV-1 p24 ELISA. (F) J1.1 cells were pretreated with indicated concentrations of GYY4137 or 5 mM NAC for 24 hr and then stimulated with 5 ng/ml PMA for 12 hr. Cells were harvested to isolate total RNA and HIV-1 reactivation was assessed by gag RT-qPCR. (G) Primary human CD4⁺ T cells purified from PBMCs samples of healthy donors were activated with anti-CD3/anti-CD28 beads for 3 days. Activated primary CD4⁺ T cells (three healthy donors) were pre-treated with 800 μM GYY4137 for 6 hr, and infected with HIV-NL4.3 (1 ng p24/10⁶ cells). Post-infection (p.i.) cells were washed, seeded in fresh media, and treated with 800 μM GYY4137 or left untreated. Virus released in the supernatant was quantified by p24 ELISA at 3^rd and 5^th day post-infection. Results are expressed as ± standard deviation and data are representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001, ns, non-significant by two-way ANOVA with Tukey’s multiple comparison test. PBMC, peripheral blood mononuclear cell; RT-qPCR, reverse transcription quantitative PCR.

Figure 4—figure supplement 1. Effect of GYY4137 on HIV-1 reactivation and cellular viability.

(A) U1 cells were pretreated with 5 mM GYY4137 or 5 mM NAC for 24 hr and then stimulated with 5 ng/ml PMA for 24 or 48 hr. Cells were stained with 3 μM propidium iodide (PI) for 15 min in dark, washed, and analyzed using flow cytometry. (B) Decomposed GYY4137 (spent GYY4137) which was aerated for at least 180 days at left at room temperature was used to pretreat U1 cells. Cells pretreated with 5 mM spent GYY4137 for 24 hr or left untreated were stimulated with 5 ng/ml PMA for 24 hr and HIV-1 reactivation was assessed by gag RT-qPCR. (C) J1.1 cells were pretreated with GYY4137 for 24 hr and then stimulated with PMA for 12 hr. Cells were then harvested, stained with PI, and subjected to flow cytometry to assess viability. (D) J-Lat cells were pretreated with 5 mM NAC or indicated concentrations of GYY4137 for 24 hr and then stimulated with 2.5 μM prostratin for 24 hr. HIV-1 reactivation was determined by estimating GFP expressing cells using flow cytometry. (E) Primary human CD4⁺ T cells purified from PBMCs samples of healthy donors were activated with CD3/CD28 for 3 days. Post-activation CD4⁺ T cells were treated with indicated concentration of GYY4137. Cell viability was determined at 3^rd day post-treatment using Live/Dead fixable far red dead cell stain kit (Invitrogen) by flow cytometry. Error bar represents standard deviations from mean. Results are representative of data from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, nonsignificant, by two-way ANOVA with Tukey’s multiple comparison test. PBMC, peripheral blood mononuclear cell; RT-qPCR, reverse transcription quantitative PCR.