Investigating the redox signaling mechanisms during HIV-TB co-infection

Abstract

Oxidative stress has been at the forefront of HIV/AIDS-related pathophysiologies since the early days of its discovery. Overcoming HIV-1 latency, wherein, the virus remains integrated in the host chromatin, undetected and unaffected by the antiretrovirals, is essential to achieve curative treatment. HIV patients display various signs of redox imbalance including low antioxidants (glutathione; GSH) and antioxidant enzymes [glutathione peroxidases (GPXs), superoxide dismutase (SOD), and catalase (CAT)], leading to an increase in reactive oxygen species (ROS). Prior study by our group supports these findings and hinted that GPXs, which is differentially regulated during latency and reactivation, could play a vital role in the HIV-1 life. However, over-expression of cellular GPXs has proved to be inconsistent as their activity is governed by different post-translational modification (PTMs), or the availability of essential micronutrient, selenium. Hence, to dissect the role of GPX during HIV-1 life cycle, our study focussed on the application of novel tools that are independent of physiological constraints (i.e., pH, mild temperature and aqueous buffers; PTMs, micronutrients). We exploited a newly emerging form of nanoparticles (NPs) which mimic enzymatic activity (nanozymes), to study the redox-based mechanisms underlying viral latency and reactivation. Part 1: Synthesis of novel morphologies of GPX-mimicking vanadium pentoxide (V2O5) nanozymes and biochemical characterisation in vitro Nanozymes are used as low-cost, efficient alternatives of natural enzymes in industries for multiple purposes, but their application in the field of biomedical research has been limited. In this context, nanozymes synthesized from vanadium pentoxide (V2O5) were shown to exhibit GPX-like activity and confer resistance against oxidative stress-mediated cell death. In this study, we synthesized and characterised a novel morphology of V2O5 nanoparticles, vanadia ultra-thin nanosheets (Vs), employing several physicochemical and biochemical techniques. Using the glutathione reductase (GR) coupled assay we established that like natural GPXs, Vs utilises GSH as a cofactor to detoxify multiple rounds of H2O2 insult. Vs followed the first order Michaelis-Menten reaction kinetics and its activity was dependent on the exposed crystal facets and surface area. Altogether, we demonstrate that Vs efficiently catalyses GSH mediated H2O2 detoxification and mimics GPX-like activity in vitro. Part 2: Assessing the antioxidant and antiviral activity of a highly efficient vanadium pentoxide (V2O5) nanozyme on HIV-1 infected cells We have previously shown that multiple isoforms of GPXs are upregulated during latent phase of HIV infection as compared to the reactivation phase. Presence of a functional, HIV-1 encoded GPX (HIV vGPX) in viral isolates from HIV-infected individuals which maintain a prolonged state of latency in contrast to their counterparts where the virus replicates actively, further enunciates the importance of GPX. Since overexpression of natural GPXs has yielded inconsistent results, we decided to employ GPX-mimicking nanozyme, Vs, to delineate the role of GPX during HIV-1 infection. Using non-invasive, real-time biosensors for H2O2 and GSH, Orp1-roGFP2 and Grx1-roGFP2, we established that Vs is an efficient GPX-mimic in monocytes with latent HIV infection. Vs treatment maintained a reducing GSH redox potential (EGSH) even after exposure to high levels of oxidative stress. Vs quenched H2O2 and subverted HIV-1 reactivation in multiple models of viral latency. In contrast to natural GPX isoforms which required selenium for their activity, the antioxidant function of Vs was independent of selenium. Importantly, Vs also displayed great potential to reduce active HIV-1 infection in both CD4+ T cells and monocytes.

Part 3: Exploring the mechanisms underlying the antiviral activity of Vs and investigating its in vivo relevance H2O2 is a key redox signaling molecule and secondary messenger, which is known to induce HIV-1 long terminal repeats (LTRs) via activation of redox-sensitive transcription factors (TFs) like NF-κB and AP-1. Using a NanoString based targeted gene expression analysis, we have shown that HIV-1 reactivation by phorbol ester, PMA is accompanied by upregulation of the enzymes involved in ROS production, redox-sensitive TFs and an overall downregulation of host antioxidant machinery. Vs pre-treatment lowers ROS inducing cellular responses, apoptotic pathways and subverts viral reactivation. Additionally, Vs curtails host factors responsible for viral packaging and release as well. We further substantiated our findings in in vivo model of HIV latency. Vs treatment of HIV transgenic mice (Tg26) significantly reduced ROS and edema (marker for oxidative stress and inflammation) in their lungs, concomitant with lowered viral transcripts. Thus, Vs treatment alters the host environment at large and keeps a check on viral reactivation. Previous reports from our laboratory has demonstrated that oxidative stress promotes synergy between HIV-1 and Mycobacterium tuberculosis (Mtb; one of the leading causes of mortality in HIV-infected individuals). Further, GSH lowers Mtb burden during HIV-TB co-infection. Here, we found that replication of different strains of Mtb - H37Rv (laboratory strain) and JAL2287 (multi-drug resistant clinical isolate) in HIV infected macrophages was completely abrogated in presence of Vs. Thus, Vs can successfully block the replication of both HIV-1 and the secondary pathogen, Mtb, reiterating the relevance of maintaining redox balance to tackle these diseases. To conclude, we have utilised a GPX-mimic to demonstrate the importance of redox signaling-based mechanisms during HIV-1 latency and reactivation. Our study accentuates that Vs can assist in strategies like ‘block and lock’ which aims to prevent HIV-1 reactivation and prolong the latent phase of HIV-1 life cycle. Altogether, we provide compelling experimental evidence to suggest that nanozymes like Vs could be helpful in devising new intervention strategies against varied infectious diseases with underlying oxidative stress associated complications.