title: Measuring glutathione redox potential of HIV-1-infected macrophages.
abstract: Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.
authors.affiliation: From the Department of Microbiology and Cell Biology, Centre for Infectious Disease and Research, Indian Institute of Sciences, Bangalore 560012.
authors.forename: Ashima
authors.initials: A
authors.lastname: Bhaskar
authors.affiliation: From the Department of Microbiology and Cell Biology, Centre for Infectious Disease and Research, Indian Institute of Sciences, Bangalore 560012, the Department of Biotechnology, Jamia Millia Islamia, New Delhi 25, India.
authors.forename: MohamedHusen
authors.initials: M
authors.lastname: Munshi
authors.affiliation: the International Centre for Genetic Engineering and Biotechnology, New Delhi 110 67, and.
authors.forename: Sohrab Zafar
authors.initials: SZ
authors.lastname: Khan
authors.affiliation: the Department of Biotechnology, Jamia Millia Islamia, New Delhi 25, India.
authors.forename: Sadaf
authors.initials: S
authors.lastname: Fatima
authors.affiliation: the International Centre for Genetic Engineering and Biotechnology, New Delhi 110 67, and.
authors.forename: Rahul
authors.initials: R
authors.lastname: Arya
authors.affiliation: the International Centre for Genetic Engineering and Biotechnology, New Delhi 110 67, and.
authors.forename: Shahid
authors.initials: S
authors.lastname: Jameel
authors.affiliation: From the Department of Microbiology and Cell Biology, Centre for Infectious Disease and Research, Indian Institute of Sciences, Bangalore 560012, asingh@mcbl.iisc.ernet.in.
authors.forename: Amit
authors.initials: A
authors.lastname: Singh
mesh_terms: D017209:Apoptosis / Q000235:genetics; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D015658:HIV Infections / Q000378:metabolism* / Q000473:pathology; D015497:HIV-1 / Q000302:isolation & purification / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D008264:Macrophages / Q000821:virology; D009169:Mycobacterium tuberculosis / Q000378:metabolism / Q000472:pathogenicity; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D020298:U937 Cells
chemical_list: D049452:Green Fluorescent Proteins; D005978:Glutathione
keywords: AIDS; Human Immunodeficiency Virus (HIV); Mycobacterium tuberculosis; Pathogenesis; Redox Signaling