Measuring Glutathione Redox Potential of HIV-1 Infected Macrophages [Molecular Bases of Disease]

November 18th, 2014 by Bhaskar, A., Munshi, M., Khan, S. Z., Fatima, S., Arya, R., Jameel, S., Singh, A.

Redox signaling plays a crucial role in the pathogenesis of Human Immunodeficiency Virus type-1 (HIV-1). Majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not inform the contributions of sub-cellular 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 (EGSH; Grx1-roGFP2) and measured sub-cellular changes in EGSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that while U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial EGSH (≈ −310 mV), active viral replication induces substantial oxidative stress (EGSH > −240 mV). Furthermore, exposure to a physiologically-relevant oxidant, hydrogen peroxide (H2O2) induces significant deviations in sub-cellular EGSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ~25 mV in EGSH 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 (Mtb) reactivate HIV-1 through modulation of intracellular EGSH. Finally, the expression analysis of U1 and patient Peripheral Blood Mononuclear Cells (PBMCs) demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.