Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis

  1. Virender Kumar Pal
  2. Ragini Agrawal
  3. Srabanti Rakshit
  4. Pooja Shekar
  5. Diwakar Tumkur Narasimha Murthy
  6. Annapurna Vyakarnam
  7. Amit Singh  Is a corresponding author
  1. Department of Microbiology and Cell Biology, Indian Institute of Science, India
  2. Centre for Infectious Disease Research (CIDR), Indian Institute of Science, India
  3. Bangalore Medical College and Research Institute, India
  4. Department of Internal Medicine, Bangalore Medical College and Research Institute, India
  5. Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, Faculty of Life Sciences & Medicine, Guy’s Hospital, King’s College London, United Kingdom

Abstract

A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.

Editor's evaluation

Pal and colleagues show that hydrogen sulfide (H2S) inhibits HIV replication and reactivation by a variety of mechanisms including inhibition of NF-κB and recruitment of the epigenetic silencer, YY1, to the HIV promoter. They further report that H2S helps in maintaining mitochondrial bioenergetics and redox homeostasis. Altogether, the study provides novel insights into the mechanisms underlying HIV transcription and reactivation.

https://doi.org/10.7554/eLife.68487.sa0

Introduction

Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), is responsible for 0.6 million deaths and 1.7 million new infections in 2019 (https://www.who.int/data/gho/data/themes/hiv-aids). Despite advances in antiretroviral therapy (ART), the persistence of latent but replication-competent HIV in cellular reservoirs is a major barrier to cure (Finzi et al., 1997; Sengupta and Siliciano, 2018; Cohn et al., 2020). Understanding host factors and signaling pathways underlying HIV latency and rebound upon cessation of ART is of the highest importance in the search for an HIV cure (Deeks et al., 2016; Collins et al., 2020). Host-generated gaseous signaling molecules such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) modulate antimicrobial activity, inflammatory response, and metabolism of immune cells to influence bacterial and viral infections (Chinta et al., 2016; Pal et al., 2018; Tinajero-Trejo et al., 2013; Rahman et al., 2020; Shiloh et al., 2008; Braverman and Stanley, 2017). Surprisingly, while circumstantial evidence links NO and CO with HIV-1 infection (Blond et al., 2000; Devadas and Dhawan, 2006; Liu et al., 2016), the role of H2S remained completely unexplored.

H2S is primarily synthesized in mammals, including humans, via the reverse transsulfuration pathway using methionine and cysteine metabolizing enzymes cystathionine-gamma-lyase (CSE/CTH), cystathionine-beta-synthase (CBS), and cysteine-aminotransferase (CAT)–3-mercaptopyruvate sulfurtransferase (MPST), respectively (Pal et al., 2018). Because H2S is lipophilic, it rapidly permeates through biological membranes without membrane channels (lipid bilayer permeability, PM≥0.5±0.4 cm/s), dissociates into HS and S2– in aqueous solution, and maintains an HS:H2S ratio of 3:1 at physiological pH (Pal et al., 2018). Interestingly, expression and enzymatic activity of CTH was diminished in human CD8+ T cells and hepatic tissues derived from AIDS patients, respectively (Hyrcza et al., 2007; Martin et al., 2001). Additionally, the levels of H2S precursors L-cysteine and methionine were severely limited in AIDS patients (Hortin et al., 1994). Also, HIV infection reduces the major cellular antioxidant glutathione (GSH) (Staal et al., 1992), whose production is dependent on L-cysteine and H2S (Kimura et al., 2010; McBean, 2012). Consequently, supplementation with N-acetyl cysteine (NAC), which is an exogenous source of L-cysteine, has been proposed for AIDS treatment (Roederer et al., 1993). Since functional CBS and CTH enzymes are known to promote T cell activation and proliferation (Miller et al., 2012), endogenous levels of H2S can potentiate T cell-based immunity against HIV. Therefore, we think that the endogenous levels and biochemical activity of H2S, which critically depends upon the cysteine and transsulfuration pathway (Mathai et al., 2009), are likely to be important determinants of HIV infection.

Calibrated production of H2S improves the functioning of various immune cells by protecting them from deleterious oxidants, stimulating mitochondrial oxidative phosphorylation (OXPHOS), and counteracting systemic inflammation (Pal et al., 2018; Modis et al., 2014; Fu et al., 2012). In contrast, supraphysiological concentrations of H2S exert cytotoxicity by inhibiting OXPHOS, promoting inflammation, and inducing redox imbalance (Pal et al., 2018; Modis et al., 2014; Fu et al., 2012). These H2S-specific physiological changes are also crucial for HIV-1 infection. First, chronic inflammation contributes to HIV-1 pathogenesis by affecting innate and adaptive immune responses (Deeks et al., 2013), impairing recovery of CD4+ T cells post-ART (Nakanjako et al., 2011), increasing the risk of comorbidities (Leng and Margolick, 2015; Alzahrani et al., 2019), and worsening organ function (Tattevin et al., 2007). Second, HIV-1 preferentially infects CD4+ T cells with high OXPHOS (Valle-Casuso et al., 2019), relies on glycolysis for continuous replication (Hegedus et al., 2014), and reactivates in response to altered mitochondrial bioenergetics (Tyagi et al., 2020). Third, a higher capacity to resist oxidative stress promotes virus latency, whereas increased oxidative stress levels induce reactivation (Bhaskar et al., 2015; Savarino et al., 2009; Shytaj et al., 2020). Fourth, H2S directly (via S-linked sulfhydration) or indirectly modulates the activity of transcription factors NF-κB and AP-1 (Sen et al., 2012; Xie et al., 2016) that are well known to trigger HIV-1 reactivation from latency (Williams et al., 2007; Brooks et al., 2003; Duverger et al., 2013). Finally, the thioredoxin/thioredoxin reductase (Trx/TrxR) system crucial for maintaining HIV-1 reservoirs (Chirullo et al., 2013) has been shown to regulate H2S-mediated S-persulfidation (Dóka et al., 2016). Altogether, many physiological processes vital for HIV-1 pathogenesis overlap with the underlying mechanism of H2S-mediated signaling.

Based on the role of H2S in regulating redox balance, mitochondrial bioenergetics, and inflammation, we hypothesize that intracellular levels of H2S modulate the HIV-1 latency and reactivation program. To test this hypothesis, we utilized cell line-based models of HIV-1 latency and CD4+ T cells of HIV-1 infected patients on suppressive ART, which harbors the latent but replication-competent virus. Biochemical and genetic approaches were exploited to investigate the link between H2S and HIV-1 latency. Finally, we used NanoString gene expression technology, oxidant-specific fluorescent dyes, and real-time extracellular flux analysis to examine the role of H2S in mediating HIV-1 persistence by regulating gene expression, redox signaling, and mitochondrial bioenergetics.

Results

Diminished biogenesis of endogenous H2S during HIV-1 reactivation

To investigate the link between HIV-1 latency and H2S, we measured changes in the expression of genes encoding H2S-generating enzymes CBS, CTH, and MPST in monocytic (U1) and lymphocytic (J1.1 and J-Lat) models of HIV-1 latency (Figure 1A). The U1 and J1.1/J-Lat cell lines are well-studied models of post-integration latency and were derived from chronically infected clones of a promonocytic (U937) and a T-lymphoid (Jurkat) cell line, respectively (Folks et al., 1987; Perez et al., 1991; Jordan et al., 2003). Both U1 and J1.1/J-Lat show very low basal expression of the HIV-1 genome, which can be induced by several latency reversal agents (LRAs) such as PMA, TNF-α, and prostratin (Figure 1B; Folks et al., 1987; Perez et al., 1991; Jordan et al., 2003). First, we confirmed virus reactivation by measuring HIV-1 gag transcript in U1 with a low concentration of PMA (5 ng/ml). Treatment of PMA induces detectable expression of gag at 12 hr, which continued to increase for the entire 36 hr duration of the experiment (Figure 1C). Next, we assessed the mRNA and protein levels of CBS, CTH, and MPST in U1 during virus latency (untreated) and reactivation (PMA-treated). The mRNA and protein expression levels of CTH showed a significant reduction at 24 and 36 hr post-PMA treatment as compared to the untreated control, whereas the expression of CBS was not detected and MPST remained unaffected (Figure 1D–E). As an uninfected control, we measured the expression of CBS, CTH, and MPST in U937 cells. In contrast to U1, PMA treatment stimulated the mRNA and protein levels of CTH in U937 (Figure 1F–G), while CBS was barely detectable. Also, we directly measured endogenous H2S levels using a fluorescent probe 7-azido-4-methylcoumarin (AzMc) that quantitatively detects H2S in living cells (Chen et al., 2013). Consistent with the expression data, reactivation of HIV-1 by PMA reduced H2S generation in U1, whereas H2S levels were significantly increased by PMA in the uninfected U937 (Figure 1H–I).

Figure 1 with 1 supplement see all
HIV-1 reactivation diminishes expression of H2S metabolic enzymes and endogenous H2S levels.

(A) Schematic showing H2S producing enzymes in mammalian cells. (B) Experimental strategy for measuring HIV-1 reactivation and H2S production in U1. (C) U1 cells were stimulated with 5 ng/ml PMA and the expression of gag transcript was measured at indicated time points. (D, E) Time-dependent changes in the expression of CTH, CBS, and MPST at mRNA and protein level during HIV-1 latency (−PMA) and reactivation (+PMA [5 ng/ml]) in U1 cells. (F, G) Time-dependent changes in the expression of CTH, CBS, and MPST at mRNA and protein level in U937 cells with or without PMA treatment. Results were quantified by densitometric analysis for CTH, CBS, and MPST band intensities and normalized to GAPDH, using ImageJ software. (H, I) U1 and U937 cells were treated with 5 ng/ml PMA for 24 hr or left untreated, stained with AzMC for 30 min at 37°C, and images were acquired using Leica TCS SP5 confocal microscope (H). Scale bar represents 40 μm. Average fluorescence intensity was quantified by ImageJ software (I). Results are expressed as mean ± standard deviation and are representative of data from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001, by two-way ANOVA with Tukey’s multiple comparison test.

Figure 1—source data 1

This file contains the source data used to generate Figure 1E.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig1-data1-v3.zip
Figure 1—source data 2

This file contains the source data used to generate Figure 1G.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig1-data2-v3.zip
Figure 1—source data 3

This file contains the source data used to make the graphs presented in Figure 1.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig1-data3-v3.xlsx

Similar to U1 monocytic cells, PMA treatment reactivated HIV-1 and reduced the expression of CTH in J1.1 lymphocytic cells but not in the uninfected Jurkat cells in a time-dependent manner (Figure 1—figure supplement 1A-B). The expression of CBS was reduced in both J1.1 and Jurkat upon PMA treatment, indicating that only CTH specifically downregulates in response to HIV-1 reactivation (Figure 1—figure supplement 1A-B). In J-Lat cells, HIV-1 genome encodes GFP, which facilitates precise measurement of PMA-mediated HIV-1 reactivation from latency by flow cytometry. Treatment with PMA reactivated HIV-1 in a time-dependent manner and diminished the expression of CBS, CTH, and MPST in J-Lat as compared to Jurkat cells (Figure 1—figure supplement 1C-D). Taken together, these data indicate that HIV-1 reactivation is associated with diminished biogenesis of endogenous H2S.

CTH-mediated reactivation of HIV-1 from latency

Our results suggest that H2S biogenesis via CTH is associated with HIV-1 latency. Therefore, we next asked whether CTH-derived H2S regulates the reactivation of HIV-1 from latency. To test this idea, we depleted endogenous CTH levels in U1 using RNA interference (RNAi). The short hairpin RNA specific for CTH (U1-shCTH) silenced the expression of CTH mRNA and protein by 90% as compared to non-targeting shRNA (U1-shNT) (Figure 2A–B and Supplementary file 1a). Moreover, using AzMC probe, we confirmed the reduction in endogenous H2S levels in U1-shCTH as compared to U1-shNT (Figure 2—figure supplement 1A-B). In addition to H2S production, CTH catalyzes the last step in the reverse transsulfuration pathway to generate cysteine. However, levels of cysteine remain comparable in U1-shCTH and U1-shNT (Figure 2—figure supplement 1C), indicating that other routes for cysteine biosynthesis (e.g., de novo and assimilatory pathways) compensate for CTH depletion in U1. Next, we investigated the effect of H2S depletion via CTH suppression on HIV-1 latency by measuring viral transcription (gag transcript), translation (HIV-1 p24 capsid protein), and release (HIV-1 p24 abundance in the cell supernatant). We found that the low basal expression of HIV-1 gag in U1-shCTH was stimulated by 16-fold upon depletion of CTH (p=0.0018) (Figure 2C). Furthermore, while PMA induced expression of gag transcript by 73-fold in U1-shNT, a further enhancement to 200-fold was observed in U1-shCTH (Figure 2C). Consistent with this, levels of p24 capsid protein inside cells or released in the supernatant were significantly elevated in U1-shCTH as compared to U1-shNT with (p=0.028) or without PMA-treatment (p=0.015) (Figure 2D–E). Deprivation of cysteine in the culture medium does not stimulate viral transcription in U1 (Figure 2—figure supplement 1D), indicating that reduction in H2S levels is likely responsible for the reactivation of HIV-1 in U1-shCTH.

Figure 2 with 1 supplement see all
Genetic silencing of CTH reactivates HIV-1 and alters gene expression associated with redox stress, apoptosis, and mitochondrial function.

(A) Total RNA was isolated from shCTH E8, shCTH E9, and non-targeting shRNA (shNT) lentiviral vectors transduced U1 cells and change in CTH mRNA was examined by RT-qPCR. (B) Cell lysates of U1, shNT, and shCTH E8 were assessed for CTH abundance using immunoblotting. The CTH band intensities were quantified by densitometric analysis and normalized to GAPDH. (C) shCTH and shNT were treated with 5 ng/ml PMA or left untreated for 24 hr and HIV-1 reactivation was determined by gag RT-qPCR. (D, E) shCTH and shNT were treated with 5 ng/ml PMA or left untreated. At the indicated time points, HIV-1 reactivation was measured by flow-cytometry using fluorescent tagged (PE-labeled) antibody specific to intracellular p24 (Gag) antigen and p24 ELISA in the supernatant. Results are expressed as mean ± standard deviation and are representative of data from two independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001, by two-way ANOVA with Tukey’s multiple comparison test. (F) Total RNA isolated from untreated or PMA (5 ng/ml; 24 hr)-treated U1-shNT and U1-shCTH were examined by NanoString Technology to assess the expression of genes associated with HIV-1 infection and oxidative stress response. Heatmap showing functional categories of significantly differentially expressed genes. Gene expression data obtained were normalized to internal control β2 microglobulin (B2M), and fold changes were calculated using the nSolver 4.0 software. Genes with fold changes values of >1.5 and p<0.05 were considered as significantly altered. RT-qPCR, reverse transcription quantitative PCR.

Figure 2—source data 1

This file contains the source data used to generate Figure 2B.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig2-data1-v3.zip
Figure 2—source data 2

This file contains the source data used to make the graphs presented in Figure 2.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig2-data2-v3.xlsx
Figure 2—source data 3

This file contains raw values associated with the NanoString analysis of host genes affected upon depletion of CTH in U1.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig2-data3-v3.xlsx

We also depleted CTH levels in J1.1 cells using RNAi and monitored HIV-1 reactivation by assessing intracellular p24 levels under basal conditions. The depletion of CTH triggered HIV-1 reactivation from latency as evident from a sevenfold increase in p24 levels as compared to shNT (Figure 2—figure supplement 1E). A minor increase in p24 levels (3.8-fold) was also apparent upon the depletion of CBS in J1.1 (Figure 2—figure supplement 1E). Finally, and consistent with U1 and J1.1, genetic silencing of CTH using a CTH specific shRNA or chemical inhibition by propargylglycine (PAG) reactivated HIV-1 in J-Lat cells (Figure 2—figure supplement 1F-I). These data indicate that CTH and likely CTH-derived H2S supports latency and impede the reactivation of HIV-1.

Altered expression of genes involved in HIV-1 reactivation upon CTH depletion

The above results indicate that CTH is a target controlling HIV-1 reactivation from latency, prompting us to investigate the mechanism. Several pathways that induce HIV-1 reactivation from latency are also influenced by H2S. These include redox signaling (Banerjee, 2011), NF-κB pathway (Gao et al., 2012), inflammatory response (Bhatia, 2012), and mitochondrial bioenergetics (Szabo et al., 2014). Hence, we conducted a focused expression profiling of 185 human genes intrinsically linked to HIV-1 reactivation using the NanoString nCounter Technology (see Supplementary file 1b for the gene list) to measure absolute amounts of multiple transcripts without reverse transcription. Because depletion of CTH promoted reactivation of the HIV-1 in U1, we compared the expression profile of U1-shCTH with U1-shNT to further understand the link between H2S biogenesis and HIV-1 latency. Examination of 84 genes related to oxidative stress response revealed differential regulation of 22 genes in U1-shCTH compared with U1-shNT (Figure 2F and Supplementary file 1c). Interestingly, more than 90% of genes showing altered expression were downregulated in U1-shCTH. Of these, genes encoding key cellular antioxidant enzymes and buffers such as catalase (CAT), superoxide dismutase (SOD1), peroxiredoxin family, thioredoxin family (TXN, TXNRD1 and TXNRD2), sulfiredoxin (SRXN1), glutathione metabolism (GCLM, GSS, and GSR), and sulfur metabolism (MPST, AHCY, and MAT2A) were significantly less expressed in U1-shCTH as compared to U1-shNT (Figure 2F). Further, while MPST was downregulated upon CTH knock-down, CBS remained undetectable in U1-shCTH and U1-shNT (Figure 2F). Since oxidative stress elicits HIV-1 reactivation (Bhaskar et al., 2015), these findings indicate that HIV-1 reactivation through CTH depletion could be a consequence of an altered redox balance. Further, pathways involved in promoting HIV-1 reactivation, such as NF-κB signaling (Williams et al., 2007) and apoptosis (Khan et al., 2015), were also induced in U1-shCTH as compared to U1-shNT (Figure 2F). Notably, multiple HIV-1 restriction factors are important for viral latency including type I interferon signaling (IRF2 and STAT-1) (Sgarbanti et al., 2002; Nguyen et al., 2018), APOBEC3G (Gillick et al., 2013), CDK9-CCNT1 ( Amini et al., 2002; Budhiraja et al., 2013), SLP-1 (McNeely et al., 1995), and chromatin remodelers (SMARCB1, BANF1, and YY1) (Rafati et al., 2011; Coull et al., 2000) were downregulated in U1-shCTH. HIV-1 proteins are known to target mitochondria to induce mitochondrial depolarization, elevate mitochondrial reactive oxygen species (ROS), and apoptosis for replication (Badley et al., 2003). Several genes involved in sustaining mitochondrial function and membrane potential (e.g., BAX, UCP2, LHPP, and MPV17) were repressed in U1-shCTH (Figure 2F), highlighting a potential association between unrestricted virus replication, mitochondrial dysfunction, and CTH depletion.

Since PMA is known to reactivate HIV-1 (Pardons et al., 2019b), we examined the gene expression in U1-shNT upon reactivation of HIV-1 by PMA. We found that 80% of genes affected by the depletion of CTH were similarly perturbed in response to PMA. Signature of transcripts associated with oxidative stress response, sulfur metabolism, anti-viral factors, and mitochondrial function was comparable in U1-shCTH and PMA-treated U1-shNT (Figure 2F). Based on these similarities, we hypothesized that combining PMA with CTH depletion would have an additive effect on the expression of genes linked to HIV-1 reactivation. Indeed, exposure of U1-shCTH to PMA induced gene expression changes, which surpassed those produced by PMA treatment or CTH-depletion alone (Figure 2F). Notably, significant expression changes in U1-shCTH upon PMA treatment are consistent with our data showing maximal HIV-1 activation under these conditions (see Figure 2C). Overall, these results indicate that CTH contributes to HIV-1 latency by modulating multiple pathways coordinating cellular homeostasis (e.g., redox balance and mitochondrial function) and the anti-viral response.

CTH is required to maintain redox homeostasis and mitochondrial function

Physiological levels of H2S support redox balance and mitochondrial function by maintaining GSH balance (Kimura et al., 2010), protecting against ROS (Kimura et al., 2010), and acting as a substrate for the electron transport chain (ETC) (Modis et al., 2014; Fu et al., 2012). On this basis, we reasoned that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction, both of which are known to promote HIV-1 reactivation (Tyagi et al., 2020; Bhaskar et al., 2015; Singh et al., 2021). We first measured total glutathione content (GSH+GSSG) and GSSG concentration in U1-shNT and U1-shCTH using chemical-enzymatic analysis of whole-cell extract (Bhaskar et al., 2015). Whole-cell glutathione content was not significantly different in U1-shNT and U1-shCTH (p=0.2801) (Figure 3A). However, the GSSG concentration was elevated in U1-shCTH resulting in a concomitant decrease in the GSH/GSSG ratio of U1-shCTH as compared to U1-shNT (p=0.0032) (Figure 3A). The increased GSSG pool and reduced GSH/GSSG poise confirm that cells are experiencing oxidative stress upon CTH depletion. We measured total ROS using a fluorescent probe, 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which non-specifically responds to any type of ROS within the cells. Both U1-shNT and U1-shCTH showed comparable levels of cytoplasmic ROS (Figure 3—figure supplement 1A), which remained unchanged after stimulation with PMA. In addition to cytoplasmic ROS, we also measured mitochondrial ROS (mitoROS) using the red fluorescent dye MitoSOX, which selectively stains mitoROS. Lowering the levels of CTH severely increased mitoROS in U1, which was further accentuated after PMA stimulation (Figure 3B). We then studied the effect of CTH depletion on mitochondrial functions using a Seahorse XF Extracellular Flux Analyzer (Agilent) as described previously by us (Figure 3C; Tyagi et al., 2020; Szabo et al., 2014). Both basal and ATP-coupled respiration was significantly decreased in U1-shCTH as compared to U1-shNT (Figure 3D–E), consistent with the role of endogenous H2S in reducing cytochrome C oxidase for respiration (Szabo et al., 2014). The maximal respiratory capacity, attained by the dissipation of the mitochondrial proton gradient with the uncoupler FCCP, was markedly diminished in U1-shCTH (Figure 3E). The maximal respiration also facilitated the estimation of the spare respiratory capacity (SRC), which was nearly exhausted in U1-shCTH. Additional hallmarks of HIV-1 reactivation and dysfunctional mitochondria such as coupling efficiency and non-mitochondrial oxygen consumption rate (nmOCR) (Tyagi et al., 2020) were also adversely affected upon depletion of CTH (Figure 3E). These results indicate that CTH depletion decelerates respiration, diminishes the capacity of macrophages to maximally respire, and promotes nmOCR. All of these parameters are important features of mitochondrial health and are likely to be crucial for maintaining HIV-1 latency.

Figure 3 with 1 supplement see all
CTH maintains redox homeostasis and mitochondrial bioenergetics to promote HIV-1 latency.

(A) Total and oxidized cellular glutathione (GSSG) content was assessed in U1-shCTH and U1-shNT cell lysates using glutathione assay kit. (B) U1-shNT and U1-shCTH were stained with MitoSOX Red dye for 30 min at 37°C and analyzed by flow cytometry (Ex-510 nm, Em-580 nm). (C) Schematic representation of Agilent Seahorse XF Cell Mito Stress test profile to assess key parameters related to mitochondrial respiration. (D) U1-shNT and U1-shCTH (5×104) were seeded in triplicate wells of XF microplate and incubated for 1 hr at 37°C in a non-CO2 incubator. Oxygen consumption was measured without adding any drug (basal respiration), followed by measurement of OCR change upon sequential addition of 1 μM oligomycin (ATP synthase inhibitor) and 0.25 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial respiration and maximizes OCR. Finally, rotenone (0.5 μM) and antimycin A (0.5 μM) were injected to completely inhibit respiration by blocking complex I and complex III, respectively. (E) Various respiratory parameters derived from OCR measurement were determined by Wave desktop software. nmOCR; non-mitochondrial oxygen consumption rate and SRC; spare respiratory capacity. Error bar represents standard deviations from mean. Results are representative of data from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-significant, by two-way ANOVA with Bonferroni’s multiple comparison test.

Figure 3—source data 1

This file contains the source data used to make the graphs presented in Figure 3.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig3-data1-v3.xlsx

A small-molecule H2S donor diminished HIV-1 reactivation and viral replication

Having shown that diminished levels of endogenous H2S are associated with redox imbalance, mitochondrial dysfunction, and reactivation of HIV-1 from latency, we next examined if elevating H2S levels using a small-molecule H2S donor- GYY4137 sustains HIV-1 latency. The GYY4137 is a widely used H2S donor that releases a low amount of H2S over a prolonged period to mimic physiological production (Li et al., 2008). Because H2S discharge is unusually sluggish by GYY4137, the final concentration of H2S released is likely to be significantly lower than the initial concentration of GYY4137. We confirmed this by measuring H2S release in U1 cells treated with low (0.3 mM) or high (5 mM) concentrations of GYY4137 using methylene blue colorimetric assay. As a control, we treated U1 cells with 0.3 mM NaHS, which rapidly releases a high amount of H2S. As expected, NaHS treatment rapidly released 90% of H2S within 5 min of treatment (Figure 4A). In contrast, GYY4137 uniformly released a low amount of H2S for the entire 120 hr duration of the experiment (Figure 4A).

Figure 4 with 1 supplement see all
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 3rd and 5th 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—source data 1

This file contains the source data used to make the graphs presented in Figure 4.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig4-data1-v3.xlsx

We systematically tested the effect of GYY4137 on HIV-1 reactivation using multiple models of HIV-1 latency and replication. As a control, we used N-acetyl cysteine (NAC) that is known to block HIV-1 reactivation (Roederer et al., 1990). Pretreatment of U1 with a non-toxic dose of GYY4137 (5 mM) (Figure 4—figure supplement 1A) diminished the expression of gag transcript by twofold at 24 hr and tenfold at 48 hr post-PMA treatment (Figure 4B). The effect of GYY4137 on p24 levels in the supernatant was even more striking as it completely abolished the time-dependent increase in p24 concentration post-PMA treatment (Figure 4C). As expected, pretreatment with NAC similarly prevented PMA-triggered reactivation of HIV-1 in U1 (Figure 4C). Pretreatment of U1 with spent GYY4137, which comprises the decomposed backbone, showed no effect on PMA induced gag transcript (Figure 4—figure supplement 1B). Because TNF-α is a physiologically relevant cytokine that reactivates HIV-1 from latency (Folks et al., 1987), we tested the effect of GYY4137 on TNF-α-mediated virus reactivation. Treatment of U1 with TNF-α stimulated the expression of gag transcript by 42-fold and 169-fold at 24 and 48 hr, post-treatment, respectively (Figure 4D). The addition of GYY4137 or NAC nearly abolished the reactivation of HIV-1 in response to TNF-α treatment (Figure 4D). Earlier, we showed that depletion of CTH stimulated HIV-1 reactivation in U1 (see Figure 2C). Therefore, we tested if GYY4137 could complement this genetic deficiency and subvert HIV-1 reactivation. The U1-shCTH cells were pre-treated with 5 mM GYY4137 and p24 levels in the supernatant were measured. The elevated levels of p24 in U1-shCTH were reduced by twofold under basal conditions and 3.2-fold upon PMA stimulation in response to GYY4137 (Figure 4E). Both RNAi and chemical complementation data provide evidence that H2S is one of the factors regulating HIV-1 latency and reactivation in U1.

Similar to U1, we next examined whether GYY4137 subverts HIV-1 reactivation in the J1.1 T-cell line model. Treatment of J1.1 with PMA for 12 hr resulted in a 38-fold increase in gag transcript as compared to untreated J1.1 (Figure 4F), indicating efficient reactivation of HIV-1. Importantly, pretreatment with various non-toxic concentrations of GYY4137 (Figure 4—figure supplement 1C) reduced the stimulation of gag transcription by twofold upon subsequent exposure to PMA (Figure 4F). Interestingly, low concentration of GYY4137 (0.3 mM) was relatively more effective than high concentration (5 mM) in subverting HIV-1 reactivation. While intriguing, these findings are consistent with the reports that high concentrations of H2S induce pro-oxidant effects on cellular physiology (Olas et al., 2019), which might have dampened the suppressive action of GYY4137 on HIV-1 transcription in J1.1. However, this needs further experimentation. The inhibitory effect of GYY4137 on HIV-1 reactivation was relatively greater compared to NAC in J1.1 (Figure 4F). Consistent with U1 and J1.1, treatment of J-Lat with 5 μM prostratin for 24 hr induced significant HIV-1 reactivation, which was translated as 100% increase in GFP+ cells (Figure 4—figure supplement 1D). Pretreatment with GYY4137 significantly reduced HIV-1 reactivation in a dose-dependent manner (Figure 4—figure supplement 1D).

We also examined if an endogenous increase in H2S by GYY4137 halts the replication of HIV-1 in primary CD4+ T cells. To this end, we infected primary CD4+ T cells isolated from peripheral blood mononuclear cells (PBMCs) of three healthy human donors with HIV-1 (pNL4.3) and monitored HIV-1 replication by measuring the levels of p24 in the supernatant at 3 and 5 days post-infection. The p24 ELISA confirmed a time-dependent increase in viral load, which was uniformly reduced upon pretreatment with a non-toxic concentration of GYY4137 (0.8 mM) (Figure 4G and Figure 4—figure supplement 1E). Overall, these data establish that elevated levels of endogenous H2S efficiently suppress HIV-1 reactivation and replication.

GYY4137 reduced the expression of host genes involved in HIV-1 reactivation

To dissect the mechanism of GYY4137-mediated inhibition on HIV-1 reactivation, we examined the expression of 185 genes associated with HIV-1 reactivation using the NanoString nCounter Technology as described above (Figure 5A and Supplementary file 1d). Expression was analyzed for viral latency (unstimulated U1), reactivation (PMA-stimulated treated U1; PMA), and H2S-mediated suppression (U1-GYY+PMA). Consistent with the role of PMA-mediated oxidative stress in preceding HIV-1 reactivation (Bhaskar et al., 2015), expression of genes encoding ROS, and RNS generating enzymes (e.g., NADPH oxidase [NCF1 and CYBB] and Nitric oxide synthase [NOS2]) were upregulated (Figure 5A). Also, the expression of major antioxidant enzymes (e.g., GPXs, PRDXs, and CAT) and redox buffers (GSH and TRX pathways) remained repressed in U1-PMA, indicative of elevated oxidative stress. Additionally, pro-inflammatory signatures (e.g., TGFB1 and SERPINA1) and trans-activators (e.g., FOS) were induced, whereas host factors involved in HIV-1 restriction (e.g., IRF1 and YY1) were repressed in U1-PMA as compared to U1. In agreement with the reduction in endogenous H2S levels during HIV-1 reactivation, expression of CTH involved in H2S anabolism was downregulated and H2S catabolism (SQRDL) was upregulated by PMA.

GYY4137 modulates Nrf2, NF-κB, and YY1 pathways.

(A) U1 cells pre-treated with 5 mM GYY4137 for 24 hr or left untreated and then stimulated with 5 ng/ml PMA for 24 hr or left unstimulated. Total RNA was isolated and expression of genes associated with HIV-1 infection and oxidative stress response was assessed by nCounter NanoString Technology. Heatmap showing functional categories of significant DEGs in all four conditions: untreated (U1), GYY4137 alone (U1-GYY) or PMA- alone (U1-PMA), and GYY4137+PMA (U1-GYY+PMA). Gene expression data obtained were normalized to internal control β2 microglobulin (B2M), and fold changes were calculated using the nSolver 4.0 software. Genes with fold changes values of >1.5 and p<0.05 were considered as significantly altered. (B) Total cell lysates were used to analyze the expression levels of Nrf2 and HMOX1 by immunoblotting. Results were quantified by densitometric analysis of Nrf2 and HMOX1 band intensities and normalized to GAPDH. (C) U1 cells were pretreated with 5 mM GYY4137 for 6 hr and then stimulated with 30 ng/ml PMA for 4 hr or left unstimulated. Cells were harvested to prepare total cell lysate. Levels of phosphorylated NF-κB p65 (Ser536), NF-κB p65, and YY1 were determined by immunoblotting. Results were quantified by densitometric analysis for each blot and were normalized to GAPDH. (D) Schematic depiction of the binding sites for NF-κB (p65-p50 heterodimer) and YY1 on HIV-1 5´-LTR. Highlighted arrow in red indicates the regions targeted for genomic qPCR; ER site for NF-κB, and RBEI and RBEIII sites for YY1 enrichments, respectively. (E, F) U1 cells were pretreated with 5 mM GYY4137 for 6 hr, stimulated with PMA (30 ng/ml) for 4 hr, fixed with formaldehyde, and lysed. Lysates were subjected to immunoprecipitation for p65 and YY1 and protein-DNA complexes were purified using protein-G magnetic beads. The enrichment of NF-κB p65 and YY1 on HIV-1 LTR was assessed by qPCR for designated regions using purified DNA as a template. Results are expressed as mean ± standard deviation and data are representative of three independent experiments *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, non-significant, by two-way ANOVA with Tukey’s multiple comparison test. DEG, differentially expressed gene.

Figure 5—source data 1

This file contains raw values associated with the NanoString analysis of host genes affected upon PMA induced HIV reactivation in presence or absence of GYY4137 treatment.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig5-data1-v3.xlsx
Figure 5—source data 2

This file contains the source data used to generate Figure 5B.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig5-data2-v3.zip
Figure 5—source data 3

This file contains the source data used to generate Figure 5C.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig5-data3-v3.zip
Figure 5—source data 4

This file contains the source data used to make the graphs presented in Figure 5.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig5-data4-v3.xlsx

We noticed that the treatment with GYY4137 reversed the effect of PMA on the expression of genes associated with oxidative stress, inflammation, anti-viral response, apoptosis, and trans-activators (Figure 5A). For example, GYY4137 elicited a robust induction of genes regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) in U1-GYY+PMA compared to U1 or U1-PMA (Figure 5A). Nrf2 acts as a master regulator of redox metabolism (Pall and Levine, 2015) by binding to the antioxidant response element (ARE) and initiating transcription of major antioxidant genes (e.g., GSH pathway, TXNRD1, HMOX1, GPX4, and SRXN1). Interestingly, while the expression of Nrf2-dependent CTH gene was induced in U1-GYY+PMA, MPST remained unaffected. The Nrf2 activity has been shown to pause HIV-1 infection by inhibiting the insertion of reverse-transcribed viral cDNA into the host chromosome (Furuya et al., 2016). Furthermore, sustained activation of Nrf2-dependent antioxidant response is essential for the establishment of viral latency (Shytaj et al., 2020). A few genes encoding H2O2 detoxifying enzymes (e.g., CAT, GPX1, and PRDXs) were repressed in U1-GYY+PMA, indicating that the expression of these enzymes was likely counterbalanced by the elevated expression of other antioxidant systems by H2S. In line with the antagonistic effect of GYY4137 on HIV-1 transcription, the expression of HIV-1 trans-activator (FOS) was downregulated, and an inhibitor of NF-κB signaling (NFKBIA) was induced in U1-GYY+PMA as compared to U1 or U1-PMA. Finally, GYY4137 stimulated the expression of several anti-HIV (YY1, STAT1, STAT3, and IRF1) and pro-survival factors (CDKN1A and LTBR) that were repressed by PMA (Figure 5A). Altogether, H2S supplementation induces a major realignment of redox metabolism and immune pathways associated with HIV-1 reactivation.

GYY4137-mediated modulation of the Keap1-Nrf2 axis, NF-κB signaling, and activity of epigenetic factor YY1

Our expression data indicate activation of the Nrf2 pathway and modulation of transcription factors such as NF-κB and YY1 upon treatment with GYY4137. We tested if the mechanism of H2S-mediated subversion of HIV-1 reactivation involves these pathways. H2S has recently been shown to prevent cellular senescence by activation of Nrf2 via S-persulfidation of its negative regulator Keap-1 (Yang et al., 2013). Under unstimulated conditions, Nrf2 binds to Keap1, and the latter promotes Nrf2 degradation via the proteasomal machinery (Yang et al., 2013). Nrf2 disassociates from the S-persulfidated form of Keap1, accumulates in the cytoplasm, and translocates to nuclei where it induces transcription of antioxidant genes upon oxidative stress (Yang et al., 2013). We first examined if GYY4137 treatment accumulates Nrf2 in the cytoplasm. As expected, Nrf2 was not detected in U1 owing to its association with Keap1 under unstimulated conditions. However, a noticeable accumulation of Nrf2 was observed in U1-GYY+PMA compared to U1 or U1-PMA (Figure 5B). As an additional verification, we quantified the levels of an Nrf2-dependent protein HMOX-1. Similar to Nrf2, the levels of HMOX-1 were also induced in U1-GYY+PMA (Figure 5B). These findings are consistent with our NanoString data showing activation of Nrf2-specific oxidative stress responsive genes in GYY4137 treated U1.

Next, we determined if GYY4137 treatment targets NF-κB, which is a major regulator of HIV-1 reactivation (Williams et al., 2007). The cellular level of phosphorylated serine-536 in p65, a major subunit of NF-κB, is commonly measured to assess NF-κB activation (Zhong et al., 1998). Since PMA reactivates HIV-1, we observed an increase in p65 ser-536 phosphorylation in U1-PMA (Figure 5C). In contrast, pretreatment with GYY4137 significantly decreased PMA-induced p65 ser-536 phosphorylation (Figure 5C). These findings suggest that GYY4137 is likely to affect the DNA binding and transcriptional activity of NF-κB. Consistent with this idea, a two-step chromatin immunoprecipitation and genomic qPCR (ChIP-qPCR) assay showed that GYY4137-treatment significantly reduced the occupancy of p65 at its binding site (ER, enhancer region) on the HIV-1 LTR (Figure 5D–E).

GYY4137 induces the expression of another transcription factor YY1, which binds to HIV-1 LTR at RBEI and RBEIII and recruits histone deacetylase (HDACs) to facilitate repressive chromatin modifications (Coull et al., 2000). Overexpression of YY1 is known to promote HIV-1 latency (Bernhard et al., 2013). We tested if GYY4137 promotes the binding of YY1 at RBEI and RBEIII sites on HIV-1 LTR by ChIP-qPCR. The occupancy of YY1 at RBEI and RBEIII was significantly enriched in the case of U1-GYY+PMA as compared to U1-PMA or U1 (Figure 5F). Taken together, these results indicate that increasing endogenous H2S levels by using a slow-releasing donor effectively modulate the Keap1-Nrf2 axis and activity of transcription factors to maintain redox balance and control HIV-1 reactivation.

GYY4137 blocks HIV-1 rebound from latent CD4+ T cells isolated from infected individuals on suppressive ART

Having shown that H2S suppresses HIV-1 reactivation in multiple cell line models of latency, we next studied the ability of GYY4137 to limit virus transcription in primary CD4+ T cells derived from virally suppressed patients. We used a previously established methodology of maintaining CD4+ T cells from infected individuals on ART for a few weeks without any loss of phenotypic characteristics associated with HIV-1 reservoirs (Kessing et al., 2017). We isolated CD4+ T cells from PBMCs of five HIV-1 infected subjects on suppressive ART. The CD4+ T cells were expanded in the presence of interleukin-2 (IL-2), phytohemagglutinin (PHA), and feeder cells (Figure 6A). The expanded cells were cultured for 4 weeks in a medium containing IL-2 with ART (100 nM efavirenz, 180 nM zidovudine, and 200 nM raltegravir) either in the presence or absence of GYY4137 (100 μM) (Figure 6A). In this model, the virus reactivates by day 14 followed by progressive suppression at day 21 and latency establishment by day 35. The virus can be reactivated from this latent phase using well-known LRAs (Kessing et al., 2017). We quantified the temporal expression of viral RNA using reverse transcription quantitative PCR (RT-qPCR) periodically at 7-day interval. The levels of viral RNA increased initially (days 7–14), followed by low to undetectable levels by day 28 (Figure 6B). By day 28, virus RNA was uniformly untraceable in cells derived from GYY4137 treated and untreated groups, indicating the establishment of latency (Figure 6B).

Figure 6 with 3 supplements see all
GYY4137 subverts HIV-1 reactivation in latent CD4+ T cells derived from HIV-1 infected patients.

(A) Schematic representation of the experimental design to study the effect of H2S on HIV-1 reactivation using CD4+ T cells from ART-treated HIV-1 infected subjects. CD4+ T cells were sorted and activated with PHA (1 μg/ml), IL-2 (100 U/ml), feeder PBMCs (gamma-irradiated) from healthy donors. CD4+ T cells were activated in the presence of ART or ART in combination with 100 μM GYY4137. Post-activation cells were cultured with ART alone or ART +GYY4137 treatment in IL-2 containing medium. (B) Total RNA was isolated from five patients CD4+ T cells expanded in ART or ART+GYY4137. HIV-1 RNA levels were measured every 7 days by ultrasensitive semi-nested RT-qPCR with detection limit of three viral RNA copies per million cells. (C) On day 28, cells were washed of any treatment and both ART or ART+GYY4137 treatment groups were stimulated with 1 μM prostratin for 24 hr. HIV-1 RNA copies were assessed by RT-qPCR. Reduction in viral stimulation in GYY4137 treated samples is represented as percentage values. ND, non-determined. (D) Aggregate plot for five patients from data shown in (C). (E, F) Primary CD4+ T cells expanded and cultured in the presence of ART or ART+GYY4137 were analyzed over time for the expression of activation (CD38) and quiescence markers (CD127) by flow cytometry. (G) Total HIV-1 DNA content was determined up to 28 days in ART or ART+GYY4137 treated groups. Results are expressed as mean ± standard deviation. **, p<0.01; ns, non-significant, by two-way ANOVA with Tukey’s multiple comparison test. RT-qPCR, reverse transcription quantitative PCR.

Figure 6—source data 1

This file contains the source data used to make the graphs presented in Figure 6.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig6-data1-v3.xlsx

We next assessed the ability of GYY4137 to efficiently block viral reactivation. On day 28, we stimulated the CD4+ T cells with the protein kinase C (PKC) activator, prostratin, in the absence of any treatment. The activation of viral transcription was measured 24 hr later by RT-qPCR. The removal of ART uniformly resulted in a viral reactivation by prostratin in CD4+ T cells of HIV1 patients (Figure 6C). In contrast, upon ART+GYY4137 removal followed by prostratin stimulation, viral reactivation was attenuated by 90% (N=5, mean) for all five patient samples, and individual inhibition ranged from 78.9% to 100% (Figure 6C). Overall, pretreatment with ART+GYY4137 significantly reduced prostratin-mediated HIV-1 reactivation when compared to ART alone (p<0.01) (Figure 6D).

We also examined the immune-phenotype of CD4+ T cells treated with ART or ART+GYY4137 by monitoring the expression of activation (CD38) and quiescence (CD127) marker. As expected, CD38 expression increased, and CD127 expression decreased at days 7 and 14 during the activation phase, followed by a gradual reversal of the pattern during the resting phase at days 21 and 28 (Figure 6E–F and Figure 6—figure supplement 1A). Interestingly, GYY4137 treatment did not alter the temporal changes in the expression of activation and quiescence markers on CD4+ T cells (Figure 6E–F). Since memory CD4+ T cells are preferentially targeted by HIV (Chomont et al., 2009; Brenchley et al., 2004), we further analyzed if the status of naive (TN), central memory (TCM), transitional memory (TTM), and effector memory (TEM) is affected by the GYY4137 in our ex vivo expansion model. We observed that the CD4+ T cells that responded to ex vivo activation were mainly composed of TTM and TEM in our patient samples (Figure 6—figure supplement 1B-E). This is consistent with the study reporting the presence of translation-competent genomes mainly in the TTM and TEM of ART-suppressed individuals (Pardons et al., 2019a). Interestingly, the fraction of TTM shows a progressive decline, whereas TEM displays gradual increase during transition from activation (7–14 days) to quiescence phase (21–28 days) (Figure 6—figure supplement 1D-E). The addition of GYY4137 did not affect dynamic changes in the frequency of TTM and TEM subpopulations (Figure 6—figure supplement 1D-E).

Finally, we tested if the reduction in viral RNA upon GYY4137 treatment is due to the loss of cells with the ability to reactivate virus or selection of cells subset that is non-responsive to reactivating stimuli. We did not find significant differences in total HIV-1 DNA content between the CD4+ T cells immediately isolated from the patient’s PBMCs (ex vivo) and the expanded cells treated with ART+ GYY4137 or ART alone for the entire duration of the experiment (Figure 6G). The viability of cells remained comparable between ART+ GYY4137 and ART treatment groups over time (Figure 6—figure supplement 1F). Moreover, we observed that prolonged treatment with GYY4137 in combination ART does not abrogate CD4+ T-cell survival and proliferative capacity in the presence of anti-CD3/anti-28 beads or Staphylococcal enterotoxin B (SEB) as T cell activation stimuli (Figure 6—figure supplement 2A-D). Additionally, GYY4137 treatment had no adverse effect on antigen (CMVpp65) specific CD4+ and CD8+ T cells activation and function upon ex vivo stimulation (Figure 6—figure supplement 2E-J and Figure 6—figure supplement 3A-B).

Altogether, using a range of cellular and immunological assays, we confirmed that the characteristics of an individual’s viral reservoir remain preserved, and the suppression of viral RNA upon GYY4137 treatment is the result of H2S-mediated inhibition of HIV-1 transcription rather than a reduction in proviral content or an altered CD4+ T cell subsets. In sum, prolonged exposure to GYY4137 results in potent inhibition of viral reactivation, suggesting a new H2S-based mechanism to neutralize bursts of virus reactivation under suppressive ART in vivo.

GYY4137 prevents mitochondrial dysfunction in CD4+ T cells of HIV-1 patients during viral rebound

Because virus reactivation upon depletion of CTH-mediated H2S generation resulted in mitochondrial dysfunction and redox imbalance in U1, we tested if the elevation of H2S levels by GYY4137 improves mitochondrial health and maintain redox homeostasis of primary CD4+ T cells during virus reactivation ex vivo. As described earlier, CD4+ T cells harboring latent virus upon prolonged (28 days) treatment of ART and ART+GYY4137 were stimulated by prostratin or left unstimulated and subjected to mitochondrial flux analysis. The unstimulated cells from both ART and ART+GYY4147 cultures did not show any difference in OCR (Figure 7A). In contrast, several features reflecting efficient mitochondrial activity such as basal respiration and ATP-coupled respiration were significantly higher in prostratin stimulated CD4+ T cells in case of ART+GYY4137 treatment than ART alone (Figure 7B–C). Consistent with this, mitoROS generation upon stimulation with prostratin or other LRAs such as PMA/ionomycin was reduced in ART+GYY4137 treated CD4+ T cells than ART alone (p=0.005 and p=0.014), and was nearly comparable to unstimulated cells (Figure 7D–E). The reduction in mitoROS could be a consequence of GYY4137-mediated increase in the expression of Nrf2-dependent antioxidant systems. We directly tested this by RT-qPCR analysis of a selected set of Nrf2-dependent genes on CD4+ T cells treated with GYY4137 for 28 days. Consistent with our findings in U1, treatment with ART+ GYY4137 uniformly induced the expression of antioxidant genes in the latently infected CD4+ T cells compared to cell treated with ART alone (Figure 7F).

Effect of H2S on mitochondrial respiration and ROS generation in latent CD4+ T cells derived from HIV-1 patients.

(A) Primary human CD4+ T cells from HIV-1 infected subjects were activated and cultured ex vivo with ART or ART+GYY4137. On day 28, cells from ART or ART+GYY4137 treatment groups were harvested to assess mitochondrial respiration by using Seahorse XF mito-stress test as described in Materials and methods. (B) Cells from ART and ART+GYY4137 treated groups were stimulated with 1 μM prostratin 6 hr. Post-stimulation mitochondrial respiration profile was determined by Seahorse XF mito-stress test. (C) Various mitochondrial respiratory parameters derived from OCR measurement were determined by Wave desktop software. nmOCR; non-mitochondrial oxygen consumption rate and SRC; spare respiratory capacity. (D) On day 28, cells from both ART and ART+GYY4137 treatment groups were stimulated with 1 μM Prostratin for 6 hr. Cells were harvested and stained with 5 μM MitoSOX-Red dye for 30 min followed by washing. Samples were analyzed by flow cytometry. Unstimulated (Uns)- cells cultured under ART alone. (E) Both ART and ART+GYY4137 treated cells at day 14 post-activation were stimulated with 1 μg/ml PMA and 100 μg/ml ionomycin (Iono) for 6 h. Cells were harvested post-stimulation and stained with 5 μM MitoSOX-Red dye. Samples were analyzed by flow cytometry to assess mitoROS generation. Unstimulated (Uns)- cells cultured under ART alone. (F) CD4+ T cells from ART and ART+GYY4137 treated groups were stimulated with 1 μM prostratin on 28th day for 24 hr or left unstimulated. Cells were harvested to isolate total RNA and expression of hmox1, txnrd1, gclc, and prdx1 were determined by RT-qPCR. Data obtained were normalized to internal control β2 microglobulin (B2M). Error bar represents standard deviations from mean. Results are representative of data from three patient samples. *, p<0.05; **, p<0.01, by two-way ANOVA with Tukey’s multiple comparison test. RT-qPCR, reverse transcription quantitative PCR.

Figure 7—source data 1

This file contains the source data used to make the graphs presented in Figure 7.

https://cdn.elifesciences.org/articles/68487/elife-68487-fig7-data1-v3.xlsx

Altogether, these data suggest that H2S not only prevents virus reactivation but also improves mitochondrial bioenergetics and maintains redox homeostasis, which could be important for in vivo suppression of viral rebound and replenishment of the reservoir.

Discussion

The major conclusion of our study is that HIV-1 reactivation is coupled to depletion of endogenous H2S, which is associated with dysfunctional mitochondrial bioenergetics, in particular suppressed OXPHOS, GSH/GSSG imbalance, and elevated mitoROS. Decreased H2S also impaired the expression of genes involved in the maintenance of redox balance, mitochondrial function, inflammation, and HIV-1 restriction, which correlates with reactivation of HIV-1. This conclusion is supported in multiple cell line models of HIV-1 latency. Chemical complementation with GYY4137 identified H2S as an effector molecule. Finally, we confirmed the clinical relevance of our finding in primary CD4+ T cells isolated from ART-suppressed patients to recapitulate features of HIV-1 latency and reactivation (Kessing et al., 2017). Overall, our data show that while H2S deficiency reactivates HIV-1, the H2S donor GYY4137 can potently inhibit residual levels of HIV-1 transcription during suppressive ART and block virus reactivation upon stimulation. Hence, our findings highlight that H2S based therapies (Szabó, 2007) can be exploited to lock HIV-1 in a state of persistent latency by impairing the ability to reactivate.

How does H2S promote HIV-1 latency and suppress reactivation? Several studies revealed that HIV-1 reactivation is associated with loss of mitochondrial functions, oxidative stress, inflammation, and apoptosis (Tyagi et al., 2020; Bhaskar et al., 2015; Khan et al., 2015; Badley et al., 2003). Several of these biological dysfunctions are corrected by H2S. For example, localized delivery of H2S in physiological concentrations improves mitochondrial respiration, mitigates oxidative stress, and exerts anti-inflammatory functions (Tinajero-Trejo et al., 2013; Kimura et al., 2010; Fu et al., 2012; Banerjee, 2011; Gao et al., 2012; Bhatia, 2012; Szabo et al., 2014; Szabó, 2007). Our NanoString findings, XF flux analysis, and redox measurement support a mechanism whereby H2S sufficiency promotes sustenance of mitochondrial health and redox homeostasis to control HIV-1 reactivation. Conversely, genetic depletion of endogenous H2S promotes HIV-1 reactivation by decelerating OXPHOS, GSH/GSSH imbalance, and elevated mitoROS. It is widely known that reduced OXPHOS leads to accumulation of NADH, which traps flavin mononucleotide (FMN) in the reduced state on respiratory complex I leading to mitoROS generation by incomplete reduction of O2 (Murphy, 2009). Also, H2S-depletion increased nmOCR, which indicates elevated activities of other ROS producing enzymes such as NADPH oxidase and cyclooxygenases/lipoxygenase (Kim et al., 2008; Chacko et al., 2014). All of these metabolic changes are likely contributors of overwhelming ROS and disruption of GSH/GSSG poise in U1-shCTH, resulting in HIV-1 reactivation. In fact, consistent with our findings, oxidative shift in GSH/GSSG poise, deregulated mitochondrial bioenergetics, and ROI has been reported to promote HIV-1 reactivation (Tyagi et al., 2020; Bhaskar et al., 2015). The addition of GYY4137 stimulated OXPHOS and blocked HIV-1 reactivation, which is in line with the known ability of H2S in sustaining respiration by acting as a substrate of cytochrome C oxidase (CcO) (Szabo et al., 2014). Also, H2S can prevent mitochondria-mediated apoptosis (Hu et al., 2009), which is crucial for HIV-1 reactivation and infection (Khan et al., 2015). Finally, activation of Nrf2-specific antioxidant pathways upon exposure to GYY4137 agrees with the potential of H2S in augmenting the cellular antioxidative defense machinery (Pal et al., 2018; Kimura et al., 2010; Banerjee, 2011). Importantly, cell lines (U1 and J1.1) modeling HIV-1 latency and PBMCs of individuals that naturally suppress HIV-1 reactivation (long-term non-progressors [LTNPs]) display robust capacities to resist oxidative stress and apoptosis (Bhaskar et al., 2015; Cohen et al., 2004).

Our transcription profiling showed a positive correlation between GYY4137-induced H2S sufficiency and HIV-1 restriction factors (e.g., YY1, APOBEC3, IRF1, and Nrf2) and negative correlation with HIV-1 trans-activators (e.g., NFKB and FOS), consistent with an interrelationship between cell metabolism and HIV-1 latency (Valle-Casuso et al., 2019; Castellano et al., 2019). Interestingly, a sustained induction of Nrf2-driven cellular antioxidant response is essential for the successful transition between productive and latent HIV-1 infection (Bhaskar et al., 2015; Shytaj et al., 2020). Moreover, inhibition of Nrf2 promoted viral transcription and increased ROS generation (Shytaj et al., 2020), whereas its activation reduced HIV-1 infection (Furuya et al., 2016). On this basis, we think that H2S-mediated inhibition of HIV-1 is partly related to the activation of Nrf2-dependent antioxidant genes. In HIV-1 infection, the suppression of viral reactivation and pro-inflammatory mediator expression was paralleled by the inhibition of NF-κB. Interestingly, H2S inhibited activation of NF-κB in several models of inflammatory diseases, including hemorrhagic shock, lung injury, and paramyxovirus infections (Cao et al., 2014; Li et al., 2015). Further, recent studies have highlighted the anti-viral and anti-inflammatory role of H2S in SARS-CoV-2 infection and envisage the possibility of including H2S donors in COVID-19 therapy (Renieris et al., 2020; Citi et al., 2020; Pozzi et al., 2021). Similarly to these findings, we found that exposure of U1 with GYY4137 inhibited the NF-κB p65 subunit and reduced its binding to HIV-1 LTR. Currently, no antiretroviral drugs inhibit basal transcription of provirus and blocks reactivation upon therapy (ART) interruption. It has been suggested that epigenetic silencing will be an important prerequisite for persistent latency (Lange et al., 2020; Kumar et al., 2015). Our expression and ChIP-qPCR data suggest that H2S could exert its influence on HIV-1 replication machinery via activating YY1- an epigenetic silencer of HIV-1 LTR (Coull et al., 2000; Bernhard et al., 2013). Interestingly, another gasotransmitter NO modulates YY1 function via S-nitrosylation of its cysteine residues (Bonavida and Baritaki, 2012), indicating that YY1 is amenable to thiol-based-modifications and raises the possibility of YY1 S-persulfidation by H2S as a mechanism to epigenetically reconfigure HIV-1/promoter LTR.

The inefficiency of current ART in preventing virus rebound after therapy cessation poses a major hurdle to HIV-1 cure. Using a primary cell system that has been successfully used to recapitulate latency and reactivation seen in patients (Kessing et al., 2017), we confirmed that pretreatment with GYY4137 attenuated viral rebound in primary human CD4+ T cells upon stimulation with prostratin. Collectively, our results suggest that H2S reduces HIV-1 transcriptional activity, promotes silencing of its promoter, and reduces its potential for reactivation. The virus-suppressing effects of H2S were observed alongside its beneficial consequences on cellular physiology (e.g., mitochondrial function and redox balance). Both mitochondrial dysfunction and oxidative stress are major complications associated with long-term exposure to ART and contribute to inflammation and organ damage (Ibeh and Emeka-Nwabunnia, 2012; Hulgan and Gerschenson, 2012; Reyskens et al., 2013). Future studies will investigate if the prolonged treatment with H2S donors results in a better outcome due to recruitment of specific repressor and/or epigenetic silencer of HIV-1 LTR without detrimental consequences on cellular physiology. In this context, it is important to carefully calibrate the concentration of H2S donors. The higher concentration of H2S is associated with toxicity and side effects (Shen et al., 2015), whereas lower micro molar concentrations are beneficial (Szabo et al., 2014). The absorption and metabolism are reasonably well characterized for exogenously administered high doses of H2S (Reiffenstein and Roth, 1992; Beauchamp et al., 1984). The sulfide oxidation pathways are involved in the oxidation of excess H2S to thiosulfate and sulfate, which are cleared from the body via the urine in free or conjugated form (Kage et al., 1997). These observations will be important for exploring future possibilities to use H2S donors in clinical settings. Importantly, several H2S donors are currently being tested in phase 2 and phase 3 human clinical trials for other indications (Wallace et al., 2020; Polhemus et al., 2015).

Blocking HIV-1 rebound by inhibitors of viral factors (e.g., Tat) invariably results in the emergence of resistant mutants (Mousseau et al., 2019). In this context, blocking HIV-1 rebounds via H2S-directed modulation of host pathways could help in overcoming the problem of evolution of escape variants. In conclusion, we identify H2S as a central factor in HIV-1 reactivation. Our systematic mechanistic dissection of the role of H2S in cellular bioenergetics, redox metabolism, and latency unifies many previous phenomena associated with viral persistence.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (H. sapiens)U1NIH HIV Reagent ProgramCat. #: ARP-165,RRID:CVCL_M769
Cell line (H. sapiens)J1.1NIH HIV Reagent ProgramCat. #: ARP-1340,RRID:CVCL_8279
Cell line (H. sapiens)ACH-2NIH HIV Reagent ProgramCat. #: ARP-349,RRID:CVCL_0138
Cell line (H. sapiens)J-Lat 6.3NIH HIV Reagent ProgramCat. #: ARP-9846,RRID:CVCL_8280
Cell line (H. sapiens)J-Lat 10.6NIH HIV Reagent ProgramCat. #: ARP-9849,RRID:CVCL_8281
Cell line (H. sapiens)TZM-blNIH HIV Reagent ProgramCat. #: ARP-8129,RRID:CVCL_B478
Cell line (H. sapiens)U937ATCCRRID:CVCL_0007
Cell line (H. sapiens)JurkatATCCRRID:CVCL_0367
Cell line (H. sapiens)HEK293TATCCRRID:CVCL_0063
AntibodyAnti-CBS (Rabbit monoclonal)AbcamCat. #: ab140600,RRID:AB_2895036WB (1:1000)
AntibodyAnti-CTH(Rabbit polyclonal)AbcamCat. #: ab151769,RRID:AB_2861405WB (1:1000)
AntibodyAnti-MPST(Rabbit polyclonal)AbcamCat. #: ab154514,RRID:AB_2895038WB (1:1000)
AntibodyAnti-HIV-1 p24(Mouse monoclonal)AbcamCat. #: ab9071,RRID:AB_306981WB (1:1000)
AntibodyAnti-NRF2 (Rabbit monoclonal)Cell Signaling TechnologyCat. #: 12721,RRID:AB_2715528WB (1:1000)
AntibodyAnti-KEAP1 (Rabbit polyclonal)Cell Signaling TechnologyCat. #: 4678,RRID:AB_10548196WB (1:1000)
AntibodyAnti-NF-kB p65(Mouse monoclonal)Cell Signaling TechnologyCat. #: 6956,RRID:AB_10828935WB (1:1000),Chromatin IP (1:50)
AntibodyAnti-Phospho-NF-kB p65(Ser536) (Rabbit monoclonal)Cell Signaling TechnologyCat. #: 3033,RRID:AB_331284WB (1:1000)
AntibodyAnti-YY1 (Rabbit monoclonal)Cell Signaling TechnologyCat. #: 63227,RRID:AB_2799641WB (1:1000),Chromatin IP (1:100)
AntibodyAnti-GAPDH (Mouse monoclonal)Cell Signaling TechnologyCat. #: 97166,RRID:AB_2756824WB (1:1000),
AntibodyAnti-rabbit IgG,HRP-linked(Goat polyclonal)Cell Signaling TechnologyCat. #: 7074,RRID:AB_2099233WB (1:10,000)
AntibodyAnti-mouse IgG,HRP-linked(Horse polyclonal)Cell Signaling TechnologyCat. #: 7076,RRID:AB_330924WB (1:10,000)
AntibodyAnti-p24, KC57-RD1 (Mouse monoclonal)Beckman CoulterCat. #: 6604667, RRID:AB_1575989Flow cytometry(1:100)
Recombinant DNA reagentPLKO.1-puro non-mammalian shRNA controlOtherKind gift fromProf. D. K. Saini
Recombinant DNA reagentpsPAX2OtherKind gift fromProf. D. K. Saini
Recombinant DNA reagentpMD2.GOtherKind gift fromProf. D. K. Saini
Recombinant DNA reagentpNL4-3NIH HIV Reagent ProgramCat. #: ARP-165
Peptide, recombinant proteinIL-2: PROLEUKINNOVARTIS
Commercial assay or kitRNeasy mini kitQIAGENCat. #: 74106
Commercial assay or kitCysteine assay kit (fluorometric)Sigma-AldrichCat. #: MAK255
Commercial assay or kitGlutathione assay kitCayman ChemicalCat. #: 703002
Commercial assay or kitSimpleChIP Enzymatic Chromatin IP KitCell Signaling TechnologyCat. #: 9003
Commercial assay or kitEasySep Human CD4+ T Cell Isolation KitSTEMCELL TechnologiesCat. #: 17952
Commercial assay or kitCellTrace Violet Cell Proliferation KitInvitrogenCat. #: C34557
Commercial assay or kitLive/Dead Fixable Aqua Dead Cell Stain KitInvitrogenCat. #: L34957
Chemical compound, drugGYY4137Sigma-AldrichCat. #: SML0100
Chemical compound, drugPMASigma-AldrichCat. #: P8139
Chemical compound, drugNACSigma-AldrichCat. #: A7250
Chemical compound, drugPAGSigma-AldrichCat. #: P7888
Chemical compound, drugPHAThermo Fisher ScientificCat. #: R30852801
Chemical compound, drugEfavirenzNIH HIV Reagent ProgramCat. #: ARP-4624
Chemical compound, drugZidovudineNIH HIV Reagent ProgramCat. #: ARP-3485
Chemical compound, drugRaltegravirNIH HIV Reagent ProgramCat. #: ARP-11680
Software, algorithmGraphPad PrismGraphPad Software(https://www.graphpad.com)RRID:SCR_002798Version 9.0.0 for Macintosh
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070
Software, algorithmWave DesktopAgilent TechnologiesRRID:SCR_014526Version 2.6
Software, algorithmnSolverNanoString TechnologiesRRID: SCR_003420Version 4.0
Software, algorithmFlowJo(https://www.flowjo.com/solutions/flowjo)RRID: SCR_0085209.9.6 and v10

Study design

Request a detailed protocol

The primary objective of this study was to understand the role H2S gas in modulating HIV-1 latency and reactivation. First, we examined the differential expression of enzymes involved in H2S biogenesis and levels of H2S upon HIV-1 latency and reactivation. Next, we genetically silenced the expression of the main H2S producing gene CTH and showed its importance in maintaining HIV-1 latency. We performed detailed mechanistic studies on understanding the role of CTH in regulating cellular antioxidant response, mitochondrial respiration, ROS generation to maintain HIV-1 latency. Finally, pharmacological donor of H2S (GYY4137) was used to reliably increase endogenous H2S levels and to study its consequence on HIV-1 latency and rebound. Primary CD4+ T cells derived from ART-treated HIV-1 infected patients were used to assess the effect of GYY4137 in modulating HIV-1 rebound by improving mitochondrial bioenergetics and mitigating redox stress.

Mammalian cell lines and culture conditions

Request a detailed protocol

The human pro-monocytic cell line U937, CD4+ T lymphocytic cell line Jurkat, HEK293T were procured from ATCC, Manassas, VA. The chronically infected U1, J1.1, ACH-2, J-Lat 6.3, J-Lat 10.6, and TZM-bl cell lines were obtained through NIH HIV Reagent Program, Division of AIDS, NIAID, NIH, USA. All cell lines are verified to be mycoplasma free by EZdetect PCR Kit for Mycoplasma Detection (HIMEDIA). The cell lines were cultured in RPMI 1640 (Cell Clone) supplemented with L-glutamine (2 mM), 10% fetal bovine serum (FBS; MP Biomedicals), penicillin (100 units/ml), streptomycin (100 μg/ml) at 37°C and 5% CO2. HEK293T and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (Cell Clone) supplemented with 10% FBS.

Chemical reagents

Request a detailed protocol

Sodium hydrosulfide (NaHS), morpholin-4-ium 4-methoxphenyl(morpholino) phosphinodithioate dichloromethane complex (GYY4137), Phorbol-12-myristate-13-acetate (PMA), N-acetyl cysteine (NAC), Prostratin, DL-Propargylglycine (PAG), and L-Cysteine were purchased from Sigma-Aldrich. Recombinant human TNF-α was purchased from InvivoGen. The antiretroviral drugs efavirenz, zidovudine, raltegravir, and lamivudine were obtained through the NIH HIV Reagent Program.

Latent viral reactivation

Request a detailed protocol

Latently infected U1, J1.1, and J-Lat 10.6 (2×105 cells/ml) were stimulated with PMA (5 ng/ml) or TNF-α (100 ng/ml) for the time indicated in the figure legends. HIV-1 reactivation was determined by intracellular gag RT-qPCR or p24 estimation in supernatant by HIV-1 p24 ELISA (J. Mitra and Co. Pvt. Ltd., India). J-Lat 6.3 cells were stimulated with Prostratin (2.5 μM) for 24 hr and HIV-1 reactivation was assessed by estimating GFP+ cells (excitation: 488 nm; emission: 510 nm) using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).

Reverse transcription quantitative PCR

Request a detailed protocol

Total cellular RNA was isolated by RNeasy Mini Kit (QIAGEN), according to the manufacturer’s protocol. RNA (500 ng) was reverse transcribed to cDNA (iScript cDNA Synthesis Kit, Bio-Rad), subjected to quantitative real-time PCR (iQ SYBR Green Supermix, Bio-Rad), and performed using the Bio-Rad C1000 real-time PCR system. HIV-1 reactivation was assessed using gene-specific primers (Supplementary file 1f). The expression level of each gene is normalized to human β-actin as an internal reference gene.

Western blot analysis

Request a detailed protocol

Total cell lysates of PMA-treated and untreated U1, J1.1, U937, and Jurkat cell lines were prepared using radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS [sodium dodecyl sulfate], 1× protease inhibitor cocktail (Sigma-Aldrich), 1× phosphatase inhibitor cocktail [Sigma-Aldrich]). After incubation on ice for 20 min, the lysates were centrifuged at 12,000 rpm, 4°C for 15 min. Clarified supernatant was taken and total protein concentration was determined by Bicinchoninic Acid Assay (Pierce, Thermo Fisher Scientific). Total protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were probed with an anti-CBS (EPR8579), CTH (ab151769), MPST (ab154514), and anti-HIV-1 p24 (ab9071) from Abcam; Nrf2 (CST-12721), Keap1 (CST-4678), NF-κB p65 (CST-6956), phospho-NF-κB p65 (Ser536) (CST-3033), YY1 (CST-63227), and GAPDH (CST-97166) from Cell Signaling Technologies, Inc; and anti-rabbit IgG (CST-7074) and anti-mouse IgG (CST-7076) were used a secondary antibody. Proteins were detected by ECL and visualized by chemiluminescence (PerkinElmer, Waltham, MA) using the Bio-Rad Chemidoc Imaging system (Hercules, CA). For membrane reprobing, stripping buffer was used (2% SDS [w/v], 62 mM Tris-Cl buffer [0.5 M, pH 6.7] and 100 mM β-mercaptoethanol) for 20 min at 55°C. After extensive washing with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma-Aldrich), membrane was blocked and reincubated with desired antibodies.

H2S detection assays

Request a detailed protocol

Endogenous H2S levels of U1 and U937 cells were detected using H2S specific fluorescent probe as described (Chen et al., 2013). Briefly, cells were treated with PMA (5 ng/ml) for 24 hr, washed with 1× PBS, and stained with 200 μM 7-Azido-4-Methylcoumarin (AzMC) (Sigma-Aldrich). The stained cells were mounted on a glass slide and visualized using Leica TCS SP5 confocal microscope (excitation: 405 nm; emission: 450 nm). Images obtained were analyzed using LAS AF Lite software (Leica Microsystems) and semi-quantification of 50 cells was performed using ImageJ software.

H2S generation was also measured using methylene blue assay. The supernatant of U1 cells treated with NaHS or GYY4137 was incubated with Zinc acetate (1%) and NaOH (3%) (1:1 ratio) to trap H2S for 30 min. The reaction was terminated using 10% trichloroacetic acid solution. Following this, reactants were incubated with 20 mM N,N-dimethylphenylendiamine (NNDPD) in 7.2 M HCl and 30 mM FeCl3 in 1.2 M HCl for 30 min and absorbance was measured at 670 nm. The concentration of H2S was determined by plotting absorbance on a standard curve generated using NaHS (0–400 μM; R2=0.9982).

Stable cell line generation

Request a detailed protocol

For generating CBS and CTH knockdown in U1 and J1.1 cells, we used validated pooled gene-specific shRNAs from the RNAi Consortium (TRC) library (Sigma-Aldrich, USA; shRNA sequences given in Supplementary file 1a). The lentiviral particles were generated in HEK293T cells using the packaging vectors, psPAX2 and pMD2.G. The pLKO.1-puro vector encoding a non-mammalian targeting shRNA (shNT) was used as a control. The U1 cells were transduced with lentiviral particles in opti-MEM containing polybrene (10 μg/ml) for 6 hr. Cells were washed and stable clones were selected in culture medium containing 250 ng/ml of puromycin. Total RNA or cell lysates were prepared to validate knockdown of CBS and CTH.

Intracellular HIV-1 P24 staining

Request a detailed protocol

For intracellular p24 staining, U1-shCTH and U1-shNT cells were stimulated with PMA (5 ng/ml), washed with PBS followed by fixation and permeabilization using a fixation and permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 50 μl of 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody (KC57-RD1; Beckman Coulter, Inc) for 30 min at room temperature. After incubation, the cells were washed two times and the fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).

Cysteine estimation

Request a detailed protocol

The quantification of cysteine was determined using Cysteine assay kit (Sigma-Aldrich). U1-shNT and U1-shCTH cells (107 cells) were harvested and lysed by sonication in assay buffer on ice. Lysates were clarified by centrifugation at 12,000 rpm, 4°C for 10 min. Then, 10 μl of sample was added to each well and incubated with enzyme mix and detection reagent as per the manufacturer’s instruction. The assay plate was read using fluorescence plate reader at Ex/Em=365/450 nm in kinetic mode at room temperature. The concentration of cysteine in sample was calculated from standard curve.

Cysteine deprivation and HIV-1 reactivation

Request a detailed protocol

U1 cells (2×105 cells/ml) were cultured in complete RPMI 1640 medium containing 208.16 μM L-cystine (as cysteine source) and 100 μM L-methionine (denoted as [+Cys]) or RPMI 1640 medium without L-methionine and L-cystine (Sigma-Aldrich, Cat #7513) supplemented with 100 μM L-methionine (Sigma-Aldrich, Cat #64319) (denoted as cysteine-free RPMI [−Cys]), 10% dialyzed FBS (Gibco), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were harvested at indicated time points and viral reactivation was analyzed by gag RT-qPCR.

NanoString nCounter assay

Request a detailed protocol

Total RNA was isolated using a RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. RNA concentration and purity were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA), bioanalyzers systems (Agilent Technologies, Inc), and Qubit assays (Thermo Fisher Scientific). An nCounter gene expression assay was performed according to the manufacturer’s protocol. The assay utilized a custom-made NanoString codeset designed to measure 185 genes, including six house-keeping genes (Supplementary file 1b). This custom-made panel included genes associated with oxidative stress and HIV-1 infection (Supplementary file 1b). All genes were assayed simultaneously in multiplexed reactions and analyzed by fully automated nCounter Prep Station and digital analyzer (NanoString Technologies). The data were normalized to B2M, used as a housekeeping gene due to its minimum % CV across the samples, and analysis was done using nSolver 4.0 software.

Measurement of oxygen consumption rates

Request a detailed protocol

OCRs were measured using a Seahorse XFp extracellular flux analyzer (Agilent Technologies) as per the manufacturer’s instructions. Briefly, cells (U1 or Primary CD4+ T cells) were seeded at a density of 104–105 per well in a Seahorse flux analyzer plate precoated with Cell-Tak (Corning). Cells were incubated for 1 hr in a non-CO2 incubator at 37°C before loading the plate in the seahorse analyzer. To assess mitochondrial respiration, three OCR measurements were performed without any inhibitor in XF assay media to measure basal respiration, followed by sequential addition of oligomycin (1 μM), an ATP synthase inhibitor (complex V), and three OCR measurements to determine ATP-linked OCR and proton leakage. Next, cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.25 μM), was injected to determine the maximal respiration rate and the SRC. Finally, rotenone (0.5 μM) and antimycin A (0.5 μM), inhibitors of NADH dehydrogenase (complex I) and cytochrome c - oxidoreductase (complex III), respectively, were injected to completely shut down the ETC to analyze nmOCR. Seahorse data were normalized to total amount of protein (μg) and mitochondrial respiration parameters were analyzed using Wave Desktop 2.6 software (Agilent Technologies).

Estimation of intracellular glutathione content and ROS

Request a detailed protocol

Total cell lysate was prepared from 107 cells using sonication in MES (2-(N-morpholino) ethanesulfonic acid) buffer. Lysates were clarified by centrifugation and total protein concentration was estimated using BCA assay. Total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured using the glutathione assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. To measure ROS, cells were loaded with 10 μM of CM-H2DCFDA (excitation: 492 nm; emission; 517 nm) or 5 μM of MitoSOX Red (excitation: 510 nm; emission; 580 nm) for 30 min at 37°C and exposed to H2O2 (100 μM) or antimycin A (2 μM) or PMA (5 ng/ml) or left untreated at 37°C and 5% CO2. The fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (BD Biosciences).

Virus production

Request a detailed protocol

HIV-1 particle production was carried out using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies), according to the manufacturer’s protocol, in HEK293T cells using HIV-1 NL4-3 DNA (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH). The medium was replaced with fresh medium at 6 hr post-transfection, and supernatants were collected after 60 hr, centrifuged (10 min, 200×g, room temperature), and filtered through a 0.45-μm-pore-size membrane filter (MDI; Membrane Technologies) to clear cell debris. Virus was concentrated using 5× PEG-it (System Biosciences) as per the manufacturer’s protocol and virus pellet obtained was aliquoted in opti-MEM and stored at –80°C. Viral titration was done using HIV-1 reporter cell line, TZM-bl (NIH HIV Reagent Program) as described earlier (Cherne et al., 2019).

Chromatin immunoprecipitation and quantitative genomic PCR

Request a detailed protocol

The chromatin immunoprecipitation (ChIP) assays were performed using SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technologies, Inc, MA) according to the manufacturer’s instructions. Briefly, 107 U1 cells were pre-treated with GYY4137 (5 mM) for 6 hr or left untreated and then stimulated with PMA (30 ng/ml) for 4 hr. Cells were fixed using 1% formaldehyde, neutralized with glycine, and harvested in ice-cold PBS. Cells were lysed and nuclei were sheared by sonication using Bioruptor Pico (Diagenode Inc) to obtain DNA fragments of 200–500 nucleotides. The clarified lysates were immunoprecipitated using ChIP-grade anti-NF-κB p65 (CST-6956) or anti-YY1 (CST-63227), or normal rabbit IgG (as a negative control) for overnight at 4°C followed by incubation with ChIP-grade protein G magnetic beads at 4°C for 4 hr. Chromatin was eluted from protein G beads, reverse-cross linked, and DNA was column purified. Quantitative genomic PCR was done by SYBR green-based real-time PCR using primers spanning the ER, RBE I, and RBE III regions on HIV-1 LTR. The GAPDH gene was used as the reference gene to see non-specific binding. Total input (10%) was used to normalize equal amount of chromatin taken across the samples. The relative proportion of co-immunoprecipitated DNA fragments were determined with the help of threshold cycle (CT) values for each qPCR product using the equation (100×2[CT(input-3.32)- CT(IP)]). The data obtained were represented as fold enrichment normalized to IgG background for each IP reaction.

Subject samples

Request a detailed protocol

PBMCs were collected from five HIV-1 seropositive subjects on stable suppressive ART and three healthy HIV-seronegative donors (Supplementary file 1e). All subject signed informed consent forms approved by the Indian Institute of Science, Bangalore and Bangalore Medical College and Research Institute (BMCRI) review boards (Institute human ethics committee [IHEC] No-3-14012020). The PBMCs isolated from blood samples using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation was used for CD4+ T cells purification. The CD4+ T cells were purified from 50×106 PBMCs using EasySep Human CD4+ T cell isolation kit (STEMCELL Technologies).

Infection of primary CD4+ T cells

Request a detailed protocol

Primary CD4+ T cells were cultured for 3 days after isolation in RPMI 1640 supplemented with 10% FBS, 100 U/ml interleukin-2 (IL-2; PROLEUKIN; NOVARTIS), and activated by adding the Dynabeads Human T-Activator CD3/CD28 using a bead/cell ratio of 1:2. Post-activation CD4+ T cells were pre-treated with 800 μM GYY4137 for 6 hr, and infected with HIV-NL4.3 (1 ng p24/ 106 cells) by spinoculation at 1000×g for 90 min at 32°C. Following infection, cells were washed and cultured in the presence or absence of 800 μM GYY4137 in complete media containing 100 U/ml IL-2. To quantify the virion release, supernatant was harvested from infected cells and centrifuged at 400×g for 10 min and virus concentration was estimated by HIV-1 p24 ELISA (J. Mitra and Co. Pvt. Ltd., India) according to the manufacturer’s instruction.

Generation of expanded primary CD4+ T cells from aviremic subjects

Request a detailed protocol

Primary CD4+ T cells from five ART suppressed, aviremic HIV-1 infected donors were cultured at 37°C in a 5% (v/v) CO2 humidified atmosphere in RPMI 1640 medium (Gibco) with GlutaMAX, HEPES, 100 U/ml IL-2, 1 μg/ml Remel phytohemagglutinin (PHA) (Thermo Fisher Scientific), gamma-irradiated feeder PBMCs (healthy control) and either ART alone (100 nM efavirenz, 180 nM zidovudine, and 200 nM raltegravir) or ART +100 μM GYY4137. After 7 days, CD4+ T cells were cultured only with ART alone or ART+GYY4137 and 100 U/ml IL-2. For stimulation experiments on day 28, ART and ART+GYY4137 were washed off and 1×106 cells were treated with 1 µM prostratin for 24 hr.

HIV-1 RNA and DNA isolation from primary CD4+ T cells

Request a detailed protocol

CD4+ T cells (1×106) from ART or ART+GYY4137 treated groups were harvested to isolate total RNA using Qiagen RNAeasy isolation kit and 200 ng of total RNA was reverse transcribed (iScript cDNA synthesis kit, Bio-Rad). Reverse transcribed cDNA was diluted tenfold and amplified using primers against HIV-1 LTRs and seminested—PCR was performed using primers and probe listed in Supplementary file 1f. Serially diluted pNL4.3 plasmid was used to obtain the standard curve. Isolation and RT-qPCR of total HIV-1 DNA were performed as described earlier (Kessing et al., 2017). Briefly, 1×106 cells were lysed (10 mM Tris-HCl, 50 nM KCl, 400 mg/ml proteinase K) at 55°C for 16 hr followed by inactivation at 95°C for 5 min. Digested product was used as a template to set up the first PCR with Taq polymerase (NEB), 1× Taq buffer, dNTPs, HIV-1 and CD3 primers for 12 cycles. The second-round amplification was done using seminested PCR strategy wherein tenfold dilution of first round PCR product was used as a template, HIV-1, and CD3 primers/probes, Taqman Fast Advance master mix (Applied Biosystems) using SetupOnePlus Real-time PCR system (Applied Biosystems). DNA isolated from ACH2 cells that contain a single copy of HIV-1 per cell was used to obtain standard curve.

Surface marker analysis of primary CD4+ T cells derived from HIV-1 patients

Request a detailed protocol

CD4+ T cells derived from HIV-1 infected patients were stained for surface markers using monoclonal antibodies: CD4-BUV395 (SK3), CD45RA-APC-H7 (HI100), CD27-BV785 (O323), CCR7-Alexa 647 (G043H7), CD38-PE-Cy5 (HIT2), and CD127-PerCP-Cy5.5 (eBioRDR5). Additionally, cells were stained with Live/Dead fixable Aqua dead cell stain or AviD (Invitrogen) to exclude dead cells from the analysis as per the manufacturer’s instructions. PBMCs from HIV positiveART-treated patients were incubated with ART or ART in combination with 100 μM GYY4137 and either left unstimulated or stimulated with either CMVpp65 (JPT Technologies) or anti-CD3/anti-CD28 beads (at a bead:cell ratio of 0.25:1) for 18 hr. Cells were stained with antibodies for analysis of cell surface markers CD3-BV570 (UCHT1), CD4-FITC (RPA-T4), CD8-PerCP-Cy5.5 (SK1), CD25-BV421 (M-A251), CD69-PE (L78), and intracellular cytokines IFNγ-Alexa Fluor 700 (B27) and IL-2-APC (MQ1-17H12). Stained samples were run on BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo version 9.9.6 and v10 (Treestar, Ashland, OR).

Cell proliferation assay

Request a detailed protocol

PHA/IL-2 expanded CD4+ T cell lines cultured for 14 days in the presence of ART or ART in combination with 100 μM GYY4137 were washed and rested overnight. Cells were labeled with 0.5 μM cell trace violet (CTV) (Invitrogen) and incubated at 37°C for 7 min in the dark, after which CTV uptake was stopped by addition of 5 ml ice-cold complete RPMI media. Cells were pelleted down washed once with complete RPMI and used in the cell proliferation assay. CTV-labeled cells were seeded at a density of 0.5×106/well and stimulated with either 1.7 μg/ml CMVpp65 (JPT Technologies), anti-CD3/anti-CD28 T cell activator beads at a bead:cell ratio of 0.25:1 (Thermo Fisher Scientific) or 100 ng/ml SEB (Sigma-Aldrich). After 3 days, IL-2 (20 U/ml) supplemented medium was added to the cells and cultured for another 2 days. Proliferation was measured by CTV dilution after 5 days by flow cytometry. In some experiments, after 5 days of culture CTV-labeled cells were pelleted and stained with antibodies to CD3, CD4, and CD25 and CD25 expression was analyzed by flow cytometry.

Statistical analysis

Request a detailed protocol

All statistical analyses were performed using GraphPad Prism software for Macintosh (version 9.0.0). The data values are indicated as mean ± S.D. For statistical analysis Student’s t-test (in which two groups are compared) and one-way or two-way ANOVA (for analysis involving multiple groups) were used. For flow cytometry experiments, data are represented as means ± SEM and p-value between paired samples was determined by Wilcoxon matched-pairs signed-rank test. Analysis of NanoString data was performed using the nSolver platform. Differences in p-values <0.05 and fold change >1.5 were considered significant.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 and 5.

References

    1. Hortin GL
    2. Landt M
    3. Powderly WG
    (1994)
    Changes in plasma amino acid concentrations in response to HIV-1 infection
    Clinical Chemistry 40:785–789.
    1. Pall ML
    2. Levine S
    (2015)
    Nrf2, a master regulator of detoxification and also antioxidant, anti-inflammatory and other cytoprotective mechanisms, is raised by health promoting factors
    Sheng Li Xue Bao: [Acta Physiologica Sinica] 10:1–18.
    1. Perez VL
    2. Rowe T
    3. Justement JS
    4. Butera ST
    5. June CH
    6. Folks TM
    (1991)
    An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation
    Journal of Immunology 147:3145–3148.

Decision letter

  1. Frank Kirchhoff
    Reviewing Editor; Ulm University Medical Center, Germany
  2. Bavesh D Kana
    Senior Editor; University of the Witwatersrand, South Africa
  3. Frank Kirchhoff
    Reviewer; Ulm University Medical Center, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, including Frank Kirchhoff as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife in the present form.

We feel that your manuscript is well performed and contains interesting data from a basic research perspective. However, as an inhibitor of HIV reactivation, H2S donors would provide clinical utility if these are more effective and safer than ART. Adding an H2S donor during ART does not really seem to be particularly useful due to viral rebound after treatment interruption. It seems that GYY4137 works essentially as a NFkB inhibitor to suppress reactivation of HIV, which also raises concerns about side-effects. Altogether, we found your findings interesting but better suitable for a more specialized journal.

Reviewer #1:

Hydrogen sulfide (H2S) is best known for it smell of rotten eggs and its high toxicity due to interference with oxygen transport. Recent studies suggest, however, that H2S is involved in various physiological and pathological processes and might exert beneficial therapeutic effects at very low doses. Thus, H2S-releasing compounds are considered for the treatment of e.g. cardiovascular diseases. In the present study, Pal et al. report that H2S also plays an important role in reactivation of HIV from latency. The first show that HIV reactivation reduces the levels of endogenous H2S in cell lines. They further show that suppression of the H2S producing enzyme CTH enhances HIV reactivation and modulates the expression of cellular genes associated with apoptosis and mitochondrial function. The authors also provide evidence that endogenous production of H2S or release from small molecule H2S donors allows U1 and Jurkat cells to maintain normal redox homeostasis and mitochondrial bioenergetics and suggest that this promotes HIV-1 latency. Finally, they provide evidence that the H2S donor GYY4137 modulates Nrf2, NF-κB, and YY1 pathways and suppresses HIV reactivation in latently infected T cells from HIV-infected individuals. The study addresses an important topic from a different angle. It is comprehensive and provides insights into the mechanisms underlying the effect of H2S on HIV latency. In addition, the impact of H2S donors on reactivation of HIV is of significant interest. Limitations of the study are that potential risks and problems associated with H2S as therapeutic agent are not addressed. H2S is highly toxic; thus, dosage will be a major challenge. Treatment would not only affect cells harboring latent HIV but all cells and the H2S-mediated mechanisms proposed to suppress HIV reactivation, such as inhibition of NF-κB and altered metabolism, would be expected to cause side-effects. Finally, the authors propose to combine H2S donors with ART to suppress HIV reactivation. However, effective ART usually prevents viral replication with high efficiency. Thus, rebound after treatment interruption (and not under ART) is the problem. In the end, a functional cure only makes sense if it doesn't need to be combined with ART and does not require daily treatment.

1. Problems of dosing and side-effects of H2S donors should be critically discussed. The advantage of combining an H2S donor with ART did not become clear to me since the latter very reliably prevents viral replication.

2. The study is comprehensive and it is appreciated that the authors analyzed the impact of an H2S donor in primary CD4+ T cells. However, all mechanistic data were obtained in cell lines that may not faithfully represent the status of latently HIV infected cells in vivo. Ideally, some key finding should be confirmed in primary cells. Otherwise this limitation should be mentioned. Similarly, the cell line used harbor copies of T cell line adapted HIV-1 strains that may differ from proviruses representing the latent HIV reservoirs in vivo.

3. The authors propose to repurpose H2S donors for clinical treatment. Are such agents already used in the clinic or currently just examined in preclinical animal models?

4. The authors state that H2S "bolsters the anti-HIV potential of immune cells". This conclusion does not seem to be fully warranted based on the data presented. Thus, the statement should be cautioned or further substantiated.

Reviewer #2:

While the idea of H2S acting as a regulator of HIV-1 latency is interesting, there is a long list of inconsistencies that hamper the enthusiasm for this manuscript.

Much of the cited literature that is used to make the case for their hypothesis is very old and actually refers to active HIV infection and patient studies prior to ART. Also, the literature they cite regarding the role of H2S as an antimicrobial agent seem to be limited to tuberculosis infection.

The choice of the latently infected model cell lines is rather unfortunate. There are much better defined models out there these days than J1.1 or U1 cells, such as the J-LAT cells from the Verdin lab or the various reporter cell lines generated by Levy and co-workers. In particularly, U1 cells should not be considered as latently infected, as the virus has a defect in the Tat/TAR axis and is mostly just transcriptionally attenuated. It is unclear why the authors only use J-LAT cells for one of the last experiments.

It is further unclear why the authors perform most of the experiments using U1 cells, which are considered promonocytic, but in the end seek to demonstrate the influence of H2S on latent HIV-1 infection in CD4 T cells. Performing all experiments in J1.1 or better J-LAT cells would have seemed more intuitive.

The authors suggest that H2S production would control latent HIV-1 infection and reactivation. Regarding the idea that CBS, CTH or possibly MPST would control latent infection as a function of their ability to produce H2S from different sources, there are several questions. First, if H2S is the primary factor, why would the presence of e.g. MPST no compensate for the reduction of CTH? Second, why would J1.1 and U1 cells both host latent HIV-1 infection events, however, their CDB/CTH/MPST composition is completely different? Third, natural variations in CTH expression caused by culture over time are larger than variations caused by PMA activation.

Also, the statement that H2S production as exerted per loss of CTH would control reactivation is not supported by the kinetic data. In latently HIV-1 infected T cell lines or monocytic cell lines, PMA-mediated HIV-1 reactivation at the protein level is usually almost complete after 24 hours, but at this time point the difference between e.g. CTH levels only begins to appear in U1 cells.

The data for J1.1. are even less convincing.

Figure 2F. PMA is known to induce an oxidative stress response, however, in the experiments the data suggest that PMA results in a downregulated oxidative stress response. Maybe the authors could explain this discrepancy with the literature. In fact, both shRNA transductions, scr and CTH-specific seem to result in a lower PMA response.

Given that the others in subsequent experiments use GYY4137, which is supposed to mimic the increased release of H2S, the authors should have definitely included experiments in which they would overexpress CTH, e.g. by retroviral transduction. Specifically in U1 cells, which seemingly do not express CBS, overexpression of CBS should also result in a suppressed phenotype.

Figure 4F: The authors need to explain how they can measure a 4-fold gag RNA expression change in untreated cells. Also, according to Figure 4A, 300 µM GYY produces much less H2S than 5mM, yet the suppressive effect of 300 µM GYY is much higher?

Initially, the authors argue "that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction to promote HIV-1 reactivation"(p. 9). Less CTH would suggest less produced H2S. However, later on in the manuscript they demonstrate that addition of a H2S source (GYY4137) results in the suppression of HIV-1 replication and supposedly HIV-1 reactivation. This is somewhat confusing.

CTH, or for that matter CBS or MPST do not only produce H2S, however, they also are part of other metabolic pathways. It would have been interesting and important to study how these metabolic pathways were affected by the genetic manipulations and also how the increased presence of H2S (GYY4137) would affect the metabolic activity of these enzymes or their expression.

H2S has been reported to cause NFkB inhibition by sulfhydration of p65; as such, the findings here are not particularly novel or surprising. Also, H2S induced sulfhydration is rather not targeted to a specific protein, let alone a HIV protein, making this approach a very unlikely alternative to current ART forms.

The description of the primary T cell model used to generate the data in Figure 6 is slightly misleading. Also, the idea of this model was originally to demonstrate that "block and lock" by didehydro-cortistatin is possible. In this application, the authors did not investigate whether GYY4137 would actually induce a HIV "block and lock" over an extended period of time.

The authors claim that the major conclusion of their study is that HIV-1 reactivation is coupled to depletion of endogenous H2S, which is associated with dysfunctional mitochondrial bioenergetics, in particular suppressed OXPHOS, GSH/GSSG imbalance, and elevated mitoROS. However, the authors never provide evidence that endogenous H2S is altered in latently HIV-1 infected cells (which may actually be an impossible task). By the end of the manuscript, the authors have not provided clear evidence that the effects of e.g. CTH deletion would be mediated by the production of H2S, and not by another function of the enzyme. Similarly, the inability of stimuli to trigger efficient HIV-1 reactivation following the provision of unnaturally high levels of H2S is not surprising given reports on the effect of GYY4137 as anti-inflammatory agent and suppressor NF-κB activation. Unless the authors were to demonstrate a true "block and lock" effect by GYY4137 the data will likely have limited impact on the HIV cure field.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for choosing to send your work entitled "Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis" for consideration at eLife. Your letter of appeal has been considered by a Senior Editor and a Reviewing editor, and we are prepared to consider a revised submission with no guarantees of acceptance.

Upon careful consideration of your letter and after discussion with reviewers, we have the following recommendations.

We recognize that there are several strong aspects of the work presented in your manuscript however, the possible clinical implications of the work are overstated. In addition, further clarification is required in terms of the molecular mechanism proposed. We are willing to consider a revised manuscript based on the following provisors:

1. Regarding the conclusion that the reactivation of HIV-2 associated with CTH knockdown must be due to an absence of H2S: This conclusion is problematic as CTH has other functions as well. In addition, other proteins can produce H2S. The addition of H2S in the form of GYY4137, and associated inhibition of HIV-1 reactivation, does not bring further clarity as the concentrations of H2S used are much higher than anything the cells could produce by themselves. As CTH catalyses the last step in the trans-sulfuration pathway from methionine to cysteine, an important experiment would be to demonstrate that CHT KO is specific for H2S production and does not affect cysteine levels. Also demonstrating that cysteine deprivation does not lead to HIV-1 reactivation due to a generalised stress response would be important.

2. Cell cultures produce so little H2S that it cannot be measured. As such, in the experiments, addition of GYY4137 produce completely unphysiological H2S concentrations. Given that protein sulfurylation affects thousands of proteins, the addition of GYY4137 is not a biologically relevant reproduction of the presence of CTH, as the experimental system may be associated proteome-wide activity changes. H2S donors are mostly considered for cancer and anti-inflammatory treatments, because they seem to be immunosuppressive. To translate these findings towards clinical relevance for a cure of latent HIV-1 infection, you would have to show that lack of reactivation is not associated with a lack of T cell response. For example, you could demonstrate that T cells, after being cultured in GYY4137 containing medium, still respond to anti-CD3 stimulation with IL-2 secretion or CD25 upregulation. A better assay would be that the extended presence of GYY4137 does not abrogate the ability of T cells to proliferate upon antigen recognition (CMV pp65 should be a good antigen in combination with CFSE stains).

3. Clinical Relevance: Unless GYY4137 produces a "lock" state of latent HIV-1 infection events, or selectively kills latently HIV-1 infected T cells, there is no clinical relevance for a cure. No one will exchange ART for an immuno-suppressive experimental treatment, that already in vitro is not sufficiently potent to completely block reactivation. We suggest you remove any focus in the discussion on possible clinical implications and approach presenting this study from a fundamental research perspective as you do have some compelling findings.

https://doi.org/10.7554/eLife.68487.sa1

Author response

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…] 1. Problems of dosing and side-effects of H2S donors should be critically discussed. The advantage of combining an H2S donor with ART did not become clear to me since the latter very reliably prevents viral replication.

We appreciate the comment. We appropriately discussed dosing and the side effects of H2S donors in the revised manuscript (Line no. 619, page no. 25 in the revised manuscript). The higher concentration of H2S is associated with toxicity and side effects (2), whereas lower micro molar concentrations are beneficial (3). The absorption and metabolism are reasonably well characterized for exogenously administered high doses of H2S (4,5). The sulfide oxidation pathways is involved in oxidation of excess H2S to thiosulphate and sulphate, which are cleared from the body via the urine in free or conjugated form (6). We assessed cytotoxicity of H2S donor (GYY4137) in all cell lines and primary CD4+ T cell model and selected non-toxic concentrations of GYY4137 to perform assays. As suggested by the editors, we have toned down the clinical potential of H2S donor in the revised manuscript.

2. The study is comprehensive and it is appreciated that the authors analyzed the impact of an H2S donor in primary CD4+ T cells. However, all mechanistic data were obtained in cell lines that may not faithfully represent the status of latently HIV infected cells in vivo. Ideally, some key finding should be confirmed in primary cells. Otherwise this limitation should be mentioned. Similarly, the cell line used harbor copies of T cell line adapted HIV-1 strains that may differ from proviruses representing the latent HIV reservoirs in vivo.

We appreciate the reviewer’s concern. For the reasons outlined by the reviewer, we examined the effect of GYY4137 on the CD4+ T cells derived from latent HIV-1 infected patients. Consistent with the cell line data, GYY4137 maintains mitochondrial bioenergetics (OXPHOS), suppresses mitochondrial reactive oxygen species (ROS), and induces the expression of the Nrf2-dependent antioxidant genes (Figure 6 and 7 in the revised manuscript). In the revised manuscript, we further extended these observations and showed that the effect of GYY4137 is not due to the impaired CD4+ T cells activation (Figure 6—figure supplement 2A-J in the revised manuscript).

To further allay the reviewer’s concern, we isolated primary CD4+ T cells from healthy donors and assessed the influence of GYY4137 on viral replication. Similar to findings in cell lines, GYY4137 diminished HIV-1 replication in primary CD4+ T cells without affecting cellular viability (Figure 4G and Figure 4—figure supplement 1E in the revised manuscript).

3. The authors propose to repurpose H2S donors for clinical treatment. Are such agents already used in the clinic or currently just examined in preclinical animal models?

Several H2S donors are in human clinical trials. A list of donors and the progress made in clinical trials are indicated in Author response table 1. We have included this information in the revised manuscript (Line no. 627, page no. 26 in the revised manuscript)

Author response table 1
List of H2S releasing donors and the progress made in clinical trials.
SI no.Name of H2S donorsCompanyCharacteristicPotential applicationDevelopment stage
1ACS-15CTG PharmaDiclofenac derivative (NSAID)ArthritisPre-clinical
2ATB-346AntibeNaproxen derivative (NSAID)OsteoarthritisPhase 3
3ATB-429AntibeNSAID-coupledInflammatory bowel diseasePre-clinical
4SG1002SulfagenixHeart failurePhase 2
5IK-1001IkariaNa2SMultiple hypoxic/ischaemic conditionPhase 2
6GYY4137Lawesson’s reagentParkinson’s diseasePre-clinical

4. The authors state that H2S "bolsters the anti-HIV potential of immune cells". This conclusion does not seem to be fully warranted based on the data presented. Thus, the statement should be cautioned or further substantiated.

We have rephrased the sentence to reflect our data showing the ability of GYY4137 to reduce the chances of viral rebound.

Reviewer #2:

While the idea of H2S acting as a regulator of HIV-1 latency is interesting, there is a long list of inconsistencies that hamper the enthusiasm for this manuscript.

Much of the cited literature that is used to make the case for their hypothesis is very old and actually refers to active HIV infection and patient studies prior to ART. Also, the literature they cite regarding the role of H2S as an antimicrobial agent seem to be limited to tuberculosis infection.

We have revised the list of literature and included more relevant references post-ART era. Recently, the antimicrobial role of H2S is comprehensively examined in the context of tuberculosis. Given the close association of TB with HIV, we thought our study is very timely and essential. However, we would like to point out that the references showing the effect of H2S on infection caused by respiratory viruses are included in the manuscript (7-9). Further, recent findings showing the influence of H2S in the context of SARS-CoV2 infection are also included in the revised manuscript (Line no. 592, page no. 24 in the revised manuscript).

The choice of the latently infected model cell lines is rather unfortunate. There are much better defined models out there these days than J1.1 or U1 cells, such as the J-LAT cells from the Verdin lab or the various reporter cell lines generated by Levy and co-workers. In particularly, U1 cells should not be considered as latently infected, as the virus has a defect in the Tat/TAR axis and is mostly just transcriptionally attenuated. It is unclear why the authors only use J-LAT cells for one of the last experiments.

As suggested by the reviewer, we have generated new data using J-LAT cells in the revised manuscript. First, we confirmed that PMA-mediated HIV-1 reactivation in J-LAT cells is associated with the down-regulation of cbs, cth, and mpst transcripts (Figure 1—figure supplement 1C-D in the revised manuscript). Additionally, we have performed several other mechanistic experiments in J-LAT cells to validate the data generated in U1 (see response below).

It is further unclear why the authors perform most of the experiments using U1 cells, which are considered promonocytic, but in the end seek to demonstrate the influence of H2S on latent HIV-1 infection in CD4 T cells. Performing all experiments in J1.1 or better J-LAT cells would have seemed more intuitive.

The choice of U1 was based on our earlier studies showing that U1 cells uniformly recapitulate the association of redox-based mechanisms and mitochondrial bioenergetics with HIV-latency and reactivation (10-12). We have validated key findings of U1 cells in J1.1 and J-Lat cell lines. We genetically and chemically silenced the expression of CTH in J-Lat cells and examined the effect on HIV-1 reactivation. Consistent with U1 and J1.1, genetic silencing of CTH using CTH-specific shRNA (shCTH) reactivated HIV-1 in J-Lat (Figure 2—figure supplement 1F-G in the revised manuscript). Supporting this, pre-treatment of J-Lat with non-toxic concentrations of a well-established CTH inhibitor, propargylglycine (PAG) further stimulated PMA-induced HIV-1 reactivation (Figure 2—figure supplement 1H-I in the revised manuscript). Altogether, using various cell line models of HIV-1 latency, we confirmed that endogenous H2S biogenesis counteracts HIV-1 reactivation.

The authors suggest that H2S production would control latent HIV-1 infection and reactivation. Regarding the idea that CBS, CTH or possibly MPST would control latent infection as a function of their ability to produce H2S from different sources, there are several questions. First, if H2S is the primary factor, why would the presence of e.g. MPST no compensate for the reduction of CTH? Second, why would J1.1 and U1 cells both host latent HIV-1 infection events, however, their CDB/CTH/MPST composition is completely different? Third, natural variations in CTH expression caused by culture over time are larger than variations caused by PMA activation.

These questions are important and complex. CBS, CTH, and MPST produce H2S in the sulfur network. CBS and CTH reside in the cytoplasm, whereas MPST is mainly involved in cysteine catabolism and is mitochondrial localized. The lack of compensation of CTH by MPST could be due to the compartmentalization of their activities. Furthermore, CTH and CBS activities are regulated by diverse metabolites, including heme, S-adenosyl methionine (SAM), and nitric oxide/carbon monoxide (NO/CO). In contrast, MPST activity responds to cysteine availability. How substrates/cofactors availability and enzyme choices are regulated in the cellular milieu of J1.1 and U1 is an interesting question for future experimentation.

Moreover, the tissue-specific expression/activity of CBS and CTH dictates their relative contributions in H2S biogenesis and cellular physiology (13). Some of these factors are likely responsible for differential expression of CBS, CTH, and MPST in J1.1 and U1 cells. Regardless of these concerns, viral reactivation uniformly reduces the expression of CTH in U1, J1.1, and J-Lat. While we cannot completely rule out natural variations in CTH expression over prolonged culturing, in our experimental setup CTH remained stably expressed and consistently showed down-regulation upon PMA treatment as compared to untreated conditions.

Also, the statement that H2S production as exerted per loss of CTH would control reactivation is not supported by the kinetic data. In latently HIV-1 infected T cell lines or monocytic cell lines, PMA-mediated HIV-1 reactivation at the protein level is usually almost complete after 24 hours, but at this time point the difference between e.g. CTH levels only begins to appear in U1 cells.

The data for J1.1. are even less convincing.

We have performed the kinetics of p24 production and CTH in U1 cells. We showed that the levels of p24 gradually increased from 6 h and kept on increasing till the last time point, i.e., 36 h post-PMA-treatment (Figure 2D in the revised manuscript). The p24 ELISA detected a similar kinetics of p24 increase in the cell supernatant (Figure 2E in the revised manuscript). The CTH levels show reduction at 24 h and 36 h. Based on these data, we report that HIV-1 reactivation is associated with diminished biogenesis of endogenous H2S. We have not made any claims that depletion of CTH precedes HIV reactivation. However, our CTH knockdown data clearly showed that diminished expression of CTH reactivates HIV-1 in the absence of PMA, which is consistent with our hypothesis that H2S production is likely to be a critical host component for maintaining viral latency.

Figure 2F. PMA is known to induce an oxidative stress response, however, in the experiments the data suggest that PMA results in a downregulated oxidative stress response. Maybe the authors could explain this discrepancy with the literature. In fact, both shRNA transductions, scr and CTH-specific seem to result in a lower PMA response.

In our experiment, PMA treatment for 24 h results in down-regulation of oxidative stress genes. However, the effect of PMA on the oxidative stress responsive genes is time-dependent. In our earlier publication, we showed that 12 h PMA treatment induces oxidative stress responsive genes in U1 cells (12), whereas at 24 h, the expression of genes is down-regulated (10). Genetic silencing of CTH resulted in elevated mitochondrial ROS and GSH imbalance, which is in line with a further decrease in the expression of oxidative stress responsive genes as compared to PMA alone. As a consequence, PMA-treatment of U1-shCTH induced HIV-1 reactivation, which supersedes that stimulated by PMA or shCTH alone.

Given that the others in subsequent experiments use GYY4137, which is supposed to mimic the increased release of H2S, the authors should have definitely included experiments in which they would overexpress CTH, e.g. by retroviral transduction. Specifically in U1 cells, which seemingly do not express CBS, overexpression of CBS should also result in a suppressed phenotype.

We have explored the role of elevated H2S levels using GY44137. Treatment with GYY4137 suppressed HIV reactivation in multiple cell lines and primary CD4+ T cells. As suggested by the reviewer, overexpression of CTH could be another strategy to validate these findings. However, since the transsulfuration pathway and active methyl cycle are interconnected and share metabolic intermediates (e.g., homocysteine), overexpression of CTH could disturb this balance and may lead to metabolic paralysis. Owing to these potential limitations, we used a slow releasing H2S donor (GYY4137) to chemically complement CTH deficiency during HIV reactivation. We thank the reviewer for this comment.

Figure 4F: The authors need to explain how they can measure a 4-fold gag RNA expression change in untreated cells. Also, according to Figure 4A, 300 µM GYY produces much less H2S than 5mM, yet the suppressive effect of 300 µM GYY is much higher?

The four-fold-expression in untreated cells is likely due to leaky control of viral transcription in J1.1 cells (14 16). However, to avoid confusion, we have replotted the results by normalizing the data generated upon PMA mediated HIV reactivation with the PMA untreated cells in the revised manuscript (Figure 4F in the revised manuscript). The suppressive effect of GYY4137 at the lower concentration is intriguing but consistent with the findings that high and low concentrations of H2S have profound and distinct effects on cellular physiology (3,17). One possibility is that the high concentration of H2S induces mitochondrial sulfide oxidation pathway to avert toxicity. This might modulate mitochondrial activity and ROS, resulting in the suppression of GYY4137 effect. Consistent with this, higher concentrations of H2S have been shown to cause pro-oxidant effects, DNA damage and genotoxicity (3,18). We have discussed these possibilities in the revised manuscript (Line no. 329, page no. 14 in the revised manuscript)

Initially, the authors argue "that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction to promote HIV-1 reactivation"(p. 9). Less CTH would suggest less produced H2S. However, later on in the manuscript they demonstrate that addition of a H2S source (GYY4137) results in the suppression of HIV-1 replication and supposedly HIV-1 reactivation. This is somewhat confusing.

We show that depletion of endogenous H2S by diminished expression of CTH (U1-shCTH) resulted in higher mitochondrial ROS and GSH/GSSG imbalance. Both of these alterations are known to reactivate HIV-1 and promote replication (10,11,19). The addition of GYY4137 chemically compensated for the diminished expression of CTH, and prevented HIV-1 reactivation in U1-shCTH. These events are expected to suppress HIV-1 replication and reactivation. We have made this distinction clear in the revised manuscript (line no. 284, page no. 12 in the revised manuscript).

CTH, or for that matter CBS or MPST do not only produce H2S, however, they also are part of other metabolic pathways. It would have been interesting and important to study how these metabolic pathways were affected by the genetic manipulations and also how the increased presence of H2S (GYY4137) would affect the metabolic activity of these enzymes or their expression.

We fully agree with the reviewer. In fact, our NanoString data show that upon CTH knockdown (U1-shCTH), MPST levels were down-regulated and CBS remained undetectable (Figure 2F in the revised manuscript). Additionally, GYY4137 treatment induced the expression of CTH but not MPST upon PMA addition (Figure 5A in the revised manuscript). We have incorporated these findings in the revised manuscript (line no. 215, page no. 9 and line no. 375, page no. 15 in the revised manuscript). Given that CBS and CTH catalyzed at least eight H2S generating steps and two cysteine-producing reactions, the modulation of CTH by HIV is likely to have a widespread influence on transsulfuration pathway and active methyl cycle intermediates. Our future strategies are to generate a comprehensive understanding of sulfur metabolism underlying HIV latency and reactivation. These experiments require multiple biochemical and genetic technologies with appropriate controls. We hope that the reviewer would agree with our views that these experiments should be a part of future investigation. We thank the reviewer for this comment.

H2S has been reported to cause NFkB inhibition by sulfhydration of p65; as such, the findings here are not particularly novel or surprising. Also, H2S induced sulfhydration is rather not targeted to a specific protein, let alone a HIV protein, making this approach a very unlikely alternative to current ART forms.

We believe that NF-κB inhibition is not the only mechanism by which H2S exerts its influence on HIV latency. Recent studies point towards the importance of the Nrf2-Keap1 axis in sustaining HIV-latency (20). Our data suggest an important role for Nrf2-Keap1 signaling in mediating the influence of H2S on HIV latency. Additionally, recruitment of an epigenetic silencer YY1 is also affected by H2S. Interestingly, YY1 activity is modulated by redox signaling (21), suggesting H2S could be an important regulator of YY1 activity in HIV-infected cells. We have so far, no evidence for viral proteins targeted by H2S. However, experiments to examine global S-persulfidation of host and HIV protein are ongoing in the laboratory to fill this knowledge gap. Lastly, our findings raise the possibility of exploring H2S donors with the current ART (not as an alternate to ART) for reducing virus reactivation. We have tone down the clinical relevance of our findings.

The description of the primary T cell model used to generate the data in Figure 6 is slightly misleading. Also, the idea of this model was originally to demonstrate that "block and lock" by didehydro-cortistatin is possible. In this application, the authors did not investigate whether GYY4137 would actually induce a HIV "block and lock" over an extended period of time.

As suggested by the reviewer, we have cited the didehydro-cortistatin studies as the basis of our strategy. Our idea was to adapt the primary T cell model to begin understanding the role of H2S in blocking HIV rebound. Our results indicate the future possibility of investigating GYY4137 to lock HIV in deep latency for an extended period of time. However, comprehensive investigation would require long-term experiments and samples from multiple HIV subjects. In the current pandemic times with overburdened Indian clinical settings, we cannot plan these experiments. However, we hope our data form a solid foundation for HIV researchers to perform extended “block and lock” studies using H2S donors.

The authors claim that the major conclusion of their study is that HIV-1 reactivation is coupled to depletion of endogenous H2S, which is associated with dysfunctional mitochondrial bioenergetics, in particular suppressed OXPHOS, GSH/GSSG imbalance, and elevated mitoROS. However, the authors never provide evidence that endogenous H2S is altered in latently HIV-1 infected cells (which may actually be an impossible task). By the end of the manuscript, the authors have not provided clear evidence that the effects of e.g. CTH deletion would be mediated by the production of H2S, and not by another function of the enzyme. Similarly, the inability of stimuli to trigger efficient HIV-1 reactivation following the provision of unnaturally high levels of H2S is not surprising given reports on the effect of GYY4137 as anti-inflammatory agent and suppressor NF-κB activation. Unless the authors were to demonstrate a true "block and lock" effect by GYY4137 the data will likely have limited impact on the HIV cure field.

It's difficult to measure H2S levels in the latently infected primary cells due to the assay's sensitivity and the insufficient number of cells latently infected with HIV-1. However, in the revised manuscript we have clearly shown that cysteine levels are not affected by CTH depletion and cysteine deprivation does not reactivate HIV-1. These results indicate that the effects of CTH depletion are likely mediated by H2S. This is consistent with our data showing that GYY4137 specifically complement CTH deficiency and blocks HIV-1 reactivation in U1-shCTH. Further, we carried in-depth investigation to show that the effect of GYY4137 is not due to impaired activation of CD4+ T cells.

Lastly, since CTH catalyzed multiple reactions during H2S production, we cannot rule out the effect of other metabolites in this process. However, we think that this is outside the scope of the present study. Our study focuses on understanding of how H2S modulates redox, mitochondrial bioenergetics, and gene expression in the context of HIV latency. These understandings are likely to positively impact future studies exploring the role of H2S on HIV cure.

References:

1. Zhu, J., Berisa, M., Schwörer, S., Qin, W., Cross, J. R., and Thompson, C. B. (2019) Transsulfuration Activity Can Support Cell Growth upon Extracellular Cysteine Limitation. Cell Metabolism 30, 865-876.e865

2. Shen, Y., Shen, Z., Luo, S., Guo, W., and Zhu, Y. Z. (2015) The Cardioprotective Effects of Hydrogen Sulfide in Heart Diseases: From Molecular Mechanisms to Therapeutic Potential. Oxid Med Cell Longev 2015, 925167

3. Szabo, C., Ransy, C., Módis, K., Andriamihaja, M., Murghes, B., Coletta, C., Olah, G., Yanagi, K., and Bouillaud, F. (2014) Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. Br J Pharmacol 171, 2099-2122

4. R J Reiffenstein, W C Hulbert, a., and Roth, S. H. (1992) Toxicology of Hydrogen Sulfide. Annual Review of Pharmacology and Toxicology 32, 109-134

5. Beauchamp, R. O., Jr., Bus, J. S., Popp, J. A., Boreiko, C. J., and Andjelkovich, D. A. (1984) A critical review of the literature on hydrogen sulfide toxicity. Crit Rev Toxicol 13, 25-97

6. Kage, S., Takekawa, K., Kurosaki, K., Imamura, T., and Kudo, K. (1997) The usefulness of thiosulfate as an indicator of hydrogen sulfide poisoning: three cases. Int J Legal Med 110, 220-222

7. Li, H., Ma, Y., Escaffre, O., Ivanciuc, T., Komaravelli, N., Kelley, J. P., Coletta, C., Szabo, C., Rockx, B., Garofalo, R. P., and Casola, A. (2015) Role of hydrogen sulfide in paramyxovirus infections. J Virol 89, 5557-5568

8. Ivanciuc, T., Sbrana, E., Ansar, M., Bazhanov, N., Szabo, C., Casola, A., and Garofalo, R. P. (2016) Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection. Am J Respir Cell Mol Biol 55, 684-696

9. Bazhanov, N., Escaffre, O., Freiberg, A. N., Garofalo, R. P., and Casola, A. (2017) Broad-Range Antiviral Activity of Hydrogen Sulfide Against Highly Pathogenic RNA Viruses. Scientific Reports 7, 41029

10. Bhaskar, A., Munshi, M., Khan, S. Z., Fatima, S., Arya, R., Jameel, S., and Singh, A. (2015) Measuring glutathione redox potential of HIV-1-infected macrophages. J Biol Chem 290, 1020-1038

11. Tyagi, P., Pal, V. K., Agrawal, R., Singh, S., Srinivasan, S., and Singh, A. (2020) Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics. mBio 11

12. Singh, S., Ghosh, S., Pal, V. K., Munshi, M., Shekar, P., Narasimha Murthy, D. T., Mugesh, G., and Singh, A. (2021) Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential. EMBO Mol Med 13, e13314

13. Liu, Y. H., Lu, M., Hu, L. F., Wong, P. T., Webb, G. D., and Bian, J. S. (2012) Hydrogen sulfide in the mammalian cardiovascular system. Antioxid Redox Signal 17, 141-185

14. Krishnan, V., and Zeichner, S. L. (2004) Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency. Journal of Virology 78, 9458-9473

15. Symons, J., Chopra, A., Malatinkova, E., De Spiegelaere, W., Leary, S., Cooper, D., Abana, C. O., Rhodes, A., Rezaei, S. D., Vandekerckhove, L., Mallal, S., Lewin, S. R., and Cameron, P. U. (2017) HIV integration sites in latently infected cell lines: evidence of ongoing replication. Retrovirology 14, 2

16. Butera, S. T., Roberts, B. D., Lam, L., Hodge, T., and Folks, T. M. (1994) Human immunodeficiency virus type 1 RNA expression by four chronically infected cell lines indicates multiple mechanisms of latency. J Virol 68, 2726-2730

17. Whiteman, M., Li, L., Rose, P., Tan, C. H., Parkinson, D. B., and Moore, P. K. (2010) The effect of hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in macrophages. Antioxid Redox Signal 12, 1147-1154

18. Olas, B., Brodek, P., and Kontek, B. (2019) The Effect of Hydrogen Sulfide on Different Parameters of Human Plasma in the Presence or Absence of Exogenous Reactive Oxygen Species. Antioxidants (Basel) 8

19. El-Amine, R., Germini, D., Zakharova, V. V., Tsfasman, T., Sheval, E. V., Louzada, R. A. N., Dupuy, C., Bilhou-Nabera, C., Hamade, A., Najjar, F., Oksenhendler, E., Lipinski, M., Chernyak, B. V., and Vassetzky, Y. S. (2018) HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production. Redox Biol 15, 97-108

20. Shytaj, I. L., Lucic, B., Forcato, M., Penzo, C., Billingsley, J., Laketa, V., Bosinger, S., Stanic, M., Gregoretti, F., Antonelli, L., Oliva, G., Frese, C. K., Trifunovic, A., Galy, B., Eibl, C., Silvestri, G., Bicciato, S., Savarino, A., and Lusic, M. (2020) Alterations of redox and iron metabolism accompany the development of HIV latency. EMBO J 39, e102209

21. Bonavida, B., and Baritaki, S. (2012) Inhibition of Epithelial-to-Mesenchymal Transition (EMT) in Cancer by Nitric Oxide: Pivotal Roles of Nitrosylation of NF-kappaB, YY1 and Snail. For Immunopathol Dis Therap 3, 125-133

[Editors’ note: what follows is the authors’ response to the second round of review.]

Upon careful consideration of your letter and after discussion with reviewers, we have the following recommendations.

We recognize that there are several strong aspects of the work presented in your manuscript however, the possible clinical implications of the work are overstated. In addition, further clarification is required in terms of the molecular mechanism proposed. We are willing to consider a revised manuscript based on the following provisors:

1. Regarding the conclusion that the reactivation of HIV-2 associated with CTH knockdown must be due to an absence of H2S: This conclusion is problematic as CTH has other functions as well. In addition, other proteins can produce H2S. The addition of H2S in the form of GYY4137, and associated inhibition of HIV-1 reactivation, does not bring further clarity as the concentrations of H2S used are much higher than anything the cells could produce by themselves. As CTH catalyses the last step in the trans-sulfuration pathway from methionine to cysteine, an important experiment would be to demonstrate that CHT KO is specific for H2S production and does not affect cysteine levels. Also demonstrating that cysteine deprivation does not lead to HIV-1 reactivation due to a generalised stress response would be important.

We thank the editors for raising these concerns. To address these comments, we measured cysteine levels in cell lysates of CTH knock down U1 cells (U1-shCTH) and compared it with the control cells (U1-shNT). The cysteine levels remain comparable in the lysates derived from both the cell lines, suggesting that CTH depletion does not affect cysteine levels (Figure 2—figure supplement 1C in the revised manuscript). The data also indicate that other routes for cysteine biosynthesis (e.g., de novo and assimilatory pathways) likely compensate for CTH depletion in U1 cells. As suggested by the reviewer, we next assessed if cysteine deprivation reactivates HIV. To deprive cells for cysteine, we used RPMI medium lacking cysteine and supplemented with dialyzed FBS (Gibcoä Cat# 26400044, see Materials and methods) as per the published protocol (1). The U1 cells were cultured in complete RPMI medium or cysteine-free RPMI medium and gag transcript was measured at 6 h, 12 h, and 24 h. The expression of gag transcript was not significantly affected upon cysteine depletion, suggesting that HIV reactivation is not stimulated by deprivation of cysteine (Figure 2—figure supplement 1D in the revised manuscript).

2. Cell cultures produce so little H2S that it cannot be measured. As such, in the experiments, addition of GYY4137 produce completely unphysiological H2S concentrations. Given that protein sulfurylation affects thousands of proteins, the addition of GYY4137 is not a biologically relevant reproduction of the presence of CTH, as the experimental system may be associated proteome-wide activity changes. H2S donors are mostly considered for cancer and anti-inflammatory treatments, because they seem to be immunosuppressive. To translate these findings towards clinical relevance for a cure of latent HIV-1 infection, you would have to show that lack of reactivation is not associated with a lack of T cell response. For example, you could demonstrate that T cells, after being cultured in GYY4137 containing medium, still respond to anti-CD3 stimulation with IL-2 secretion or CD25 upregulation. A better assay would be that the extended presence of GYY4137 does not abrogate the ability of T cells to proliferate upon antigen recognition (CMV pp65 should be a good antigen in combination with CFSE stains).

This is an excellent recommendation. Accordingly, we cultured PHA/IL-2 expanded CD4+ T cell lines for 14 days in presence of ART or ART in combination with 100 μM GYY4137. Cells were then labelled with 0.5 μM cell trace violet (CTV) and stimulated with either CMV, anti-CD3/anti-CD28 beads (at a bead: cell ratio of 1:0.25) or Staphylococcal enterotoxin B (SEB;100 ng/ml). After three days, IL-2 (20 U/ml) supplemented medium was added to the cells and cultured for another two days. Proliferation was measured by CTV dilution and CD25 expression using flow cytometry after 5 days of culture (Figure 6—figure supplement 2A-D in the revised manuscript). We included activation with SEB, as it is a known superantigen that can trigger polyclonal T cell activation by binding to the T cell receptor at the variable region of the β-chain (relevant to our system). Hence, in these long-term polyclonal cultures where frequencies of T cells specific to any given antigen e.g., CMV may hugely vary between samples or CMV-specific cells may not be detected as shown by our data, we chose to more directly test CD4+ T cell survival and proliferative capacity using SEB and CD3/28 beads as stimuli. The data demonstrate that long-term culture of cells in the presence of GYY4137 does not reduce or interfere with the capacity of the cells to respond to activation by CD3/28 or in response to SEB (Figure 6—figure supplement 2A-D in the revised manuscript).

To extend the suggestion of the reviewer further, we conducted another series of experiments to test the impact of GYY4137 in a more physiologically relevant short term T cell activation assay. Here PBMCs from HIV+ ART-treated patients were incubated with ART or ART in combination with 100 μM GYY4137 for 18 hr. Cells were analyzed for surface expression of CD25 and CD69 and intracellular staining of IFNγ by flow cytometry. The comprehensive gating strategy and representative flow cytometry plots are depicted in Figure 6—figure supplement 3A-3B of the revised manuscript. The data generated demonstrate no difference between GYY4137-treated versus untreated cultures in terms of CD4+ T cell or CD8+ T cell response to CMV peptide stimulation as measured by IFNγ expression or in the capacity of CD4+ and CD8+ T cells to express CD25 or CD69 in response to either CD3/28 or CMV stimulation (Figure 6—figure supplement 2E-J in the revised manuscript). Collectively, these findings demonstrate that CD4+ T cell function in both short-term and long-term cultures is preserved in the presence of GYY4137.

3. Clinical Relevance: Unless GYY4137 produces a "lock" state of latent HIV-1 infection events, or selectively kills latently HIV-1 infected T cells, there is no clinical relevance for a cure. No one will exchange ART for an immuno-suppressive experimental treatment, that already in vitro is not sufficiently potent to completely block reactivation. We suggest you remove any focus in the discussion on possible clinical implications and approach presenting this study from a fundamental research perspective as you do have some compelling findings.

We agree with the reviewer. We have removed the statement indicating possible clinical implications of these findings.

https://doi.org/10.7554/eLife.68487.sa2

Article and author information

Author details

  1. Virender Kumar Pal

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
    2. Centre for Infectious Disease Research (CIDR), Indian Institute of Science, Bangalore, India
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8378-1823
  2. Ragini Agrawal

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
    2. Centre for Infectious Disease Research (CIDR), Indian Institute of Science, Bangalore, India
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  3. Srabanti Rakshit

    Centre for Infectious Disease Research (CIDR), Indian Institute of Science, Bangalore, India
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Pooja Shekar

    Bangalore Medical College and Research Institute, Bangalore, India
    Contribution
    Investigation, Resources
    Competing interests
    No competing interests declared
  5. Diwakar Tumkur Narasimha Murthy

    Department of Internal Medicine, Bangalore Medical College and Research Institute, Bangalore, India
    Contribution
    Investigation, Resources
    Competing interests
    No competing interests declared
  6. Annapurna Vyakarnam

    1. Centre for Infectious Disease Research (CIDR), Indian Institute of Science, Bangalore, India
    2. Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, Faculty of Life Sciences & Medicine, Guy’s Hospital, King’s College London, London, United Kingdom
    Contribution
    Formal analysis, Investigation, Methodology, Resources, Writing – review and editing
    Competing interests
    No competing interests declared
  7. Amit Singh

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
    2. Centre for Infectious Disease Research (CIDR), Indian Institute of Science, Bangalore, India
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing - original draft, Writing – review and editing
    For correspondence
    asingh@iisc.ac.in
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6761-1664

Funding

The Wellcome Trust DBT India Alliance (IA/S/16/2/502700)

  • Amit Singh

Department of Biotechnology, Ministry of Science and Technology, India (BT/PR13522/COE/34/27/2015 BT/PR29098/Med/29/1324/2018 and BT/HRD/NBA/39/07/2018-19)

  • Amit Singh

Department of Biotechnology, Ministry of Science and Technology, India (22-0905-0006-05-987 436)

  • Amit Singh

Infosys Foundation (CIDR)

  • Amit Singh

Department of Science and Technology (DST-FIST)

  • Amit Singh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors are grateful to Prof. C Grundner at the Seattle Children’s Research Institute for critical reading of the manuscript and valuable input. The authors acknowledge Prof. D K Saini at Department of Molecular Reproduction, Development and Genetics, IISc for providing shRNA constructs used in this study. The authors are grateful to Ms. S Kumar and Dr. N R Sundaresan at Department of Microbiology and Cell Biology, IISc for helping with ChIP experiments carried out in this study. The authors gratefully acknowledge the NanoString services provided by TheraCUES Innovations Pvt Ltd, Bangalore. This work was supported by Wellcome Trust-Department of Biotechnology (DBT) India Alliance grant IA/S/16/2/502700 (AS) and in part by DBT grants BT/PR13522/COE/34/27/2015, BT/PR29098/Med/29/1324/2018, and BT/HRD/NBA/39/07/2018–19 (AS), DBT-IISc Partnership Program grant 22-0905-0006-05-987 436, Infosys Foundation, and DST-FIST. AS is a senior fellow of Wellcome Trust-DBT India Alliance. VKP is grateful to Indian Institute of Science for fellowship.

Ethics

Peripheral blood mononuclear cells (PBMCs) were collected from five aviremic HIV-seropositive subjects on stable suppressive ART. All subjects provided signed informed consent approved by the Indian Institute of Science, and Bangalore Medical College and Research Institute review boards (IHEC No.- 3-14012020).

Senior Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Reviewing Editor

  1. Frank Kirchhoff, Ulm University Medical Center, Germany

Reviewer

  1. Frank Kirchhoff, Ulm University Medical Center, Germany

Publication history

  1. Received: March 17, 2021
  2. Preprint posted: April 21, 2021 (view preprint)
  3. Accepted: November 17, 2021
  4. Accepted Manuscript published: November 18, 2021 (version 1)
  5. Version of Record published: December 9, 2021 (version 2)
  6. Version of Record updated: January 10, 2022 (version 3)

Copyright

© 2021, Pal et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,493
    Page views
  • 178
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Virender Kumar Pal
  2. Ragini Agrawal
  3. Srabanti Rakshit
  4. Pooja Shekar
  5. Diwakar Tumkur Narasimha Murthy
  6. Annapurna Vyakarnam
  7. Amit Singh
(2021)
Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis
eLife 10:e68487.
https://doi.org/10.7554/eLife.68487

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Morgane Boone et al.
    Research Advance Updated

    In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Florian Bleffert et al.
    Research Article Updated

    Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.