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Hydrogen Sulfide Blocks HIV Rebound by Maintaining Mitochondrial Bioenergetics and Redox Homeostasis

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Hydrogen Sulfide Blocks HIV Rebound by Maintaining Mitochondrial Bioenergetics and Redox Homeostasis bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial 2 bioenergetics and redox homeostasis 3 4 Virender Kumar Pal1,2, Ragini Agrawal1,2, Srabanti Rakshit2, Pooja Shekar3, Diwakar 5 Tumkur Narasimha Murthy4, Annapurna Vyakarnam2, and Amit Singh*1,2. 6 7 1Department of MiCrobiology and Cell Biology, Indian Institute of SCience, Bangalore, 8 India 9 2Centre for InfeCtious Disease Research (CIDR), Indian Institute of SCience, Bangalore, 10 India 11 3Bangalore MediCal College and Research Institute, Bangalore, India 12 4Department of Internal MediCine, Bangalore MediCal College and Research Institute, 13 Bangalore, India 14 15 * Correspondence: 16 Amit Singh ([email protected]) 17 18 19 20 21 22 23 24 25 26 27 28 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 29 Abstract 30 A fundamental Challenge in HIV eradiCation is to understand how the virus establishes 31 latency, maintains stable Cellular reservoirs, and promotes rebound upon interruption 32 of antiretroviral treatment (ART). Here, we disCovered an unexpeCted role of the 33 ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reaCtivation. We 34 show that reaCtivation of HIV-1 is associated with down-regulation of the key H2S 35 producing enzyme Cystathionine-g-lyase (CTH) and reduction in endogenous H2S. 36 GenetiC silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, 37 and remodels the transCriptome of latent Cells to trigger HIV reaCtivation. ChemiCal 38 Complementation of CTH aCtivity using a slow-releasing H2S donor, GYY4137, 39 suppressed HIV reaCtivation and diminished virus repliCation. MeChanistiCally, 40 GYY4137 blocked HIV reaCtivation by inducing the Keap1-Nrf2 pathway, inhibiting 41 NF-kB, and reCruiting the epigenetiC silencer, YY1, to the HIV promoter. In latently 42 infeCted CD4+ T cells from ART-suppressed human subjeCts, GYY4137 in 43 Combination with ART prevented viral rebound and improved mitochondrial 44 bioenergetiCs. Moreover, prolonged exposure to GYY4137 exhibited no adverse 45 influence on proviral Content or CD4+ T cell subsets, indiCating that diminished viral 46 rebound is due to a loss of transCription rather than a seleCtive loss of infeCted Cells. In 47 summary, this work provides meChanistiC insight into H2S-mediated suppression of 48 viral rebound and suggests the inclusion of an H2S donor in the Current ART regimen 49 to aChieve a functional HIV-1 cure. 50 51 52 53 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 54 Introduction 55 Human ImmunodefiCiency Virus (HIV), the Causative agent of ACquired Immuno- 56 DefiCiency Syndrome (AIDS), is responsible for 0.6 million deaths and 1.7 million new 57 infeCtions in 2019 (https://www.who.int/gho/hiv/epidemic_status/deaths_text/en/). Despite 58 advances in antiretroviral therapy (ART), the persistence of latent but repliCation- 59 competent HIV in Cellular reservoirs is a major barrier to Cure (1). Understanding host 60 faCtors and signaling pathways underlying HIV latency and rebound upon Cessation of 61 ART is of the highest importance in the search for an HIV Cure (2). Host-generated 62 gaseous signaling moleCules such as nitriC oxide (NO), Carbon monoxide (CO), and 63 hydrogen sulfide (H2S) modulate antimiCrobial aCtivity, inflammatory response, and 64 metabolism of immune Cells to influence baCterial and viral infeCtions (3-8). 65 Surprisingly, while Circumstantial evidence links NO and CO with HIV-1 infeCtion (9- 66 11), the role of H2S remained Completely unexplored. 67 H2S is primarily synthesized in mammals, including humans, via the reverse 68 transsulfuration pathway using methionine and Cysteine metabolizing enzymes 69 Cystathionine-gamma-lyase (CSE/CTH), Cystathionine-beta-synthase (CBS), and 70 Cysteine-aminotransferase (CAT)-3-mercaptopyruvate sulfurtransferase (MPST), 71 respeCtively (4). BeCause H2S is lipophiliC, it rapidly permeates through biologiCal 72 membranes without membrane Channels (lipid bilayer permeability, PM ³ 0.5 ± 0.4 – 2– – 73 cm/s), dissociates into HS and S in aqueous solution, and maintains an HS : H2S ratio 74 of 3:1 at physiologiCal pH (4). Interestingly, expression and enzymatiC aCtivity of CTH 75 was diminished in human CD8+ T cells and hepatiC tissues derived from AIDS patients, 76 respeCtively (12, 13). Additionally, the levels of H2S preCursors L-Cysteine and 77 methionine were severely limited in AIDS patients (14). Also, HIV infeCtion reduces 78 the major Cellular antioxidant glutathione (GSH) (15), whose production is dependent 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 79 on L-Cysteine and H2S (16, 17). Consequently, supplementation with N-aCetyl Cysteine 80 (NAC), whiCh is an exogenous source of L-Cysteine, has been proposed for AIDS 81 treatment (18). Since functional CBS and CTH enzymes are known to promote T cell 82 aCtivation and proliferation (19), endogenous levels of H2S Can potentiate T cell-based 83 immunity against HIV. Therefore, we think that the endogenous levels and biochemiCal 84 aCtivity of H2S, whiCh CritiCally depends upon the Cysteine and transsulfuration 85 pathway (20), are likely to be important determinants of HIV infeCtion. 86 Calibrated production of H2S improves the functioning of various immune-cells 87 by proteCting from deleterious oxidants, stimulating mitochondrial oxidative 88 phosphorylation (OXPHOS), and CounteraCting systemiC inflammation (4, 21, 22). In 89 Contrast, supraphysiologiCal Concentrations of H2S exert CytotoxiCity by inhibiting 90 OXPHOS, promoting inflammation, and inducing redox imbalance (4, 21, 22). These 91 H2S-speCifiC physiologiCal Changes are also Crucial for HIV infeCtion. First, ChroniC 92 inflammation Contributes to HIV-1 pathogenesis by affeCting innate and adaptive 93 immune responses (23), impairing reCovery of CD4+ T Cells post-ART (24), increasing 94 the risk of Comorbidities (25, 26), and worsening organ function (27). SeCond, HIV-1 95 preferentially infeCts CD4+ T Cells with high OXPHOS (28), relies on glycolysis for 96 Continuous repliCation (29), and reaCtivates in response to altered mitochondrial 97 bioenergetiCs (30). Third, a higher CapaCity to resist oxidative stress promotes virus 98 latency, whereas increased oxidative stress levels induce reaCtivation (31-33). Fourth, 99 H2S direCtly (via S-linked sulfhydration) or indireCtly modulates the aCtivity of 100 transCription faCtors NF-kB and AP-1 (34, 35) that are well known to trigger HIV-1 101 reaCtivation from latency (36-38). Lastly, the thioredoxin/thioredoxin reductase 102 (Trx/TrxR) system Crucial for maintaining HIV-1 reservoirs (39) has been shown to 103 regulate H2S- mediated S-persulfidation (40). Altogether, many physiologiCal 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 104 processes vital for HIV pathogenesis overlap with the underlying meChanism of H2S 105 mediated-signaling. 106 Based on the role of H2S in regulating redox balance, mitochondrial 107 bioenergetiCs, and inflammation, we hypothesize that intraCellular levels of H2S 108 modulate the HIV-1 latency and reaCtivation program. To test this hypothesis, we 109 utilized Cell line based models of HIV-1 latency and CD4+ T cells of HIV-infeCted 110 patients on suppressive ART, whiCh harbors the latent but repliCation-Competent virus. 111 BiochemiCal and genetiC approaChes were exploited to investigate the link between H2S 112 and HIV-1 latency. Finally, we used NanoString gene expression teChnology, oxidant- 113 speCifiC fluoresCent dyes, and real-time extraCellular flux analysis to examine the role 114 of H2S in mediating HIV-1 persistence by regulating gene-expression, redox signaling, 115 and mitochondrial bioenergetiCs. 116 117 Results 118 Diminished biogenesis of endogenous H2S during HIV-1 reactivation 119 To investigate the link between HIV-1 latency and H2S, we measured Changes 120 in the expression of genes encoding H2S-generating enzymes CBS, CTH, and MPST in 121 monocytiC (U1) and lymphocytiC (J1.1) models of HIV-1 latency (Figure 1A). The U1 122 and J1.1 Cell lines are well-studied models of post-integration latency and were derived 123 from ChroniCally infeCted Clones of a promonocytiC (U937) and a T-lymphoid (Jurkat) 124 Cell line, respeCtively (41, 42). Both U1 and J1.1 show very low basal expression of the 125 HIV-1 genome, whiCh Can be induced by several latency reversal agents (LRAs) such 126 as PMA, TNF-a, and prostratin (Figure 1B) (41, 42). First, we Confirmed virus 127 reaCtivation by measuring HIV-1 gag transCript in U1 with a low Concentration of PMA 128 (5 ng/ml). Treatment of PMA induces deteCtable expression of gag at 12 h, whiCh 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.21.440760; this version posted April 21, 2021.
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