Reply to Kulic, Z. Comment on "Subhadra et al. Significant Broad-Spectrum Antiviral Activity of Bi121 against Different Variants of SARS-CoV-2. Viruses 2023, 15, 1299".

We would like to thank Dr. Zarko Kulic [...].

Significant Broad-Spectrum Antiviral Activity of Bi121 against Different Variants of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far infected 762 million people with over 6.9 million deaths worldwide. Broad-spectrum viral inhibitors that block the initial stages of infection by reducing virus binding and proliferation, thereby reducing disease severities, are still an unmet global medical need. We studied Bi121, which is a standardized polyphenolic-rich compound isolated from Pelargonium sidoides, against recombinant vesicular stomatitis virus (rVSV)-pseudotyped SARS-CoV-2S (mutations in the spike protein) of six different variants of SARS-CoV-2. Bi121 was effective at neutralizing all six rVSV-ΔG-SARS-CoV-2S variants. The antiviral activity of Bi121 was also assessed against SARS-CoV-2 variants (USA WA1/2020, Hongkong/VM20001061/2020, B.1.167.2 (Delta), and Omicron) in Vero cells and HEK-ACE2 cell lines using RT-qPCR and plaque assays. Bi121 showed significant antiviral activity against all the four SARS-CoV-2 variants tested, suggesting a broad-spectrum activity. Bi121 fractions generated using HPLC showed antiviral activity in three fractions out of eight against SARS-CoV-2. The dominant compound identified in all three fractions using LC/MS/MS analysis was Neoilludin B. In silico structural modeling studies with Neoilludin B showed that it has a novel RNA-intercalating activity toward RNA viruses. In silico findings and the antiviral activity of this compound against several SARS-CoV-2 variants support further evaluation as a potential treatment of COVID-19.

Reliable Fluorometric Detection of SARS-CoV-2 by Targeting the G-Quadruplex through pH-Triggered Conformational Polymorphism.

Unravelling unique molecular targets specific to viruses is challenging yet critical for diagnosing emerging viral diseases. Nucleic acids and proteins are the major targets in diagnostic assays of viral pathogens. Identification of novel sequences and conformations of nucleic acids as targets is desirable for developing diagnostic assays specific to a virus of interest. Here, we disclose the identification and characterization of a highly conserved antiparallel G-quadruplex (GQ)-forming DNA sequence present within the SARS-CoV-2 genome. The two-quartet GQ with unique loop compositions formed a distinct recognition motif. Design, synthesis, and fine tuning of structure-activity of a set of small molecules led to the identification of a benzobisthiazole-based fluorogenic probe which unambiguously recognizes the target SARS-CoV-2 GQ DNA. A robust cost-effective assay was developed through thermal cycler PCR-based amplification of the antiparallel GQ-forming ORF1ab region of the SARS-CoV-2 genome and endpoint fluorescence detection with the probe. An exclusive pH window (3.5-4) helped trigger reliable conformational polymorphism (RCP) involving DNA duplex to GQ transformation, which aided the development of a GQ-RCP platform for the diagnosis of SARS-CoV-2 clinical samples. This general strategy can be adapted for the development of specific diagnostic assays targeting different noncanonical nucleic acid sequences.

Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis.

A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.

Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential.

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 (V2 O5 ) 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 V2 O5 nanosheets catalyze ROS neutralization in HIV-1-infected cells and uniformly block viral reactivation and replication. Mechanistically, V2 O5 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 V2 O5 nanosheets with a pharmacological inhibitor of NF-κB (BAY11-7082) abrogated reactivation of HIV-1. Lastly, V2 O5 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 V2 O5 nanosheets against HIV and suggested nanozymes as future platforms to develop interventions against infectious diseases.

Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics.

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant-N-acetyl cysteine (NAC)-or with inhibitors of host factors-galectins and Hsp90-attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

Hydrogen sulfide in physiology and pathogenesis of bacteria and viruses.

An increasing number of studies have established hydrogen sulfide (H2 S) gas as a major cytoprotectant and redox modulator. Following its discovery, H2 S has been found to have pleiotropic effects on physiology and human health. H2 S acts as a gasotransmitter and exerts its influence on gastrointestinal, neuronal, cardiovascular, respiratory, renal, and hepatic systems. Recent discoveries have clearly indicated the importance of H2 S in regulating vasorelaxation, angiogenesis, apoptosis, ageing, and metabolism. Contrary to studies in higher organisms, the role of H2 S in the pathophysiology of infectious agents such as bacteria and viruses has been less studied. Bacterial and viral infections are often accompanied by changes in the redox physiology of both the host and the pathogen. Emerging studies indicate that bacterial-derived H2 S constitutes a defense system against antibiotics and oxidative stress. The H2 S signaling pathway also seems to interfere with redox-based events affected on infection with viruses. This review aims to summarize recent advances on the emerging role of H2 S gas in the bacterial physiology and viral infections. Such studies have opened up new research avenues exploiting H2 S as a potential therapeutic intervention. © 2018 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(5):393-410, 2018.