Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity.

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

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.

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.