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Significance For several decades, retroviral core uncoating has been thought to occur in the cytoplasm in coordination with reverse transcription, and while some recent studies have concluded that HIV-1 uncoating occurs at the nuclear envelope during nuclear import, none have concluded that uncoating occurs in the nucleus. Here, we developed methods to study HIV-1 uncoating by direct labeling and quantification of the viral capsid protein associated with infectious viral cores that produced transcriptionally active proviruses. We find that infectious viral cores in the nuclei of infected cells are largely intact and uncoat near their integration sites just before integration. These unexpected findings fundamentally change our understanding of HIV-1 postentry replication events., HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat

Reactive oxygen species (ROS) regulates the replication of human immunodeficiency virus (HIV‐1) during infection. However, the application of this knowledge to develop therapeutic strategies remained unsuccessful due to the harmful consequences of manipulating cellular antioxidant systems. Here, we show that vanadium pentoxide (V2O5) nanosheets functionally mimic natural glutathione peroxidase activity to mitigate ROS associated with HIV‐1 infection without adversely affecting cellular physiology. Using genetic reporters of glutathione redox potential and hydrogen peroxide, we showed that V2O5 nanosheets catalyze ROS neutralization in HIV‐1‐infected cells and uniformly block viral reactivation and replication. Mechanistically, V2O5 nanosheets suppressed HIV‐1 by affecting the expression of pathways coordinating redox balance, virus transactivation (e.g., NF‐κB), inflammation, and apoptosis. Importantly, a combination of V2O5 nanosheets with a pharmacological inhibitor of NF‐κB (BAY11‐7082) abrogated reactivation of HIV‐1. Lastly, V2O5 nanosheets inhibit viral reactivation upon prostratin stimulation of latently infected CD4+ T cells from HIV‐infected patients receiving suppressive antiretroviral therapy. Our data successfully revealed the usefulness of V2O5 nanosheets against HIV and suggested nanozymes as future platforms to develop interventions against infectious diseases., This study describes a vanadium pentoxide (V2O5)‐based nanozyme that bolsters the anti‐HIV potential of immune cells and suppresses viral rebound in latently infected CD4+ T cells derived from HIV subjects.

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H(2)S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (C(max) and AUC(last)) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

Background: Abnormal glutathione poise has been associated with HIV-1 infection; however, the underlying importance is unclear. Results: Measurement of glutathione-redox potential (EGSH) revealed higher capacity of latently infected cells to resist oxidative stress and apoptosis, whereas HIV-1 replication perturbed glutathione homeostasis. Conclusion: Dynamic changes in EGSH regulate HIV-1 persistence and reactivation. Significance: Glutathione-redox signaling plays a critical role in HIV-1 infection., 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 (EGSH; Grx1-roGFP2) and measured subcellular changes in EGSH 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 EGSH (approximately −310 mV), active viral replication induces substantial oxidative stress (EGSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular 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 reactivate HIV-1 through modulation of intracellular EGSH. 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.