HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters.

The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.

SARS-CoV-2 Nsp6 damages Drosophila heart and mouse cardiomyocytes through MGA/MAX complex-mediated increased glycolysis.

SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection. Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1). Complementing transcriptomic data from the fly heart reveal that this interaction blocks the antagonistic MGA/MAX complex, which shifts the balance towards MYC/MAX and activates glycolysis-with similar findings in mouse cardiomyocytes. Further, the Nsp6-induced glycolysis disrupts cardiac mitochondrial function, known to increase reactive oxygen species (ROS) in heart failure; this could explain COVID-19-associated cardiac pathology. Inhibiting the glycolysis pathway by 2-deoxy-D-glucose (2DG) treatment attenuates the Nsp6-induced cardiac phenotype in flies and mice. These findings point to glycolysis as a potential pharmacological target for treating COVID-19-associated heart failure.

The Effects of Increased Glucose Level and Glycolysis on SARS CoV-2 Infection.

COVID-19 has entered our lives as an infection with high mortality rates. Although the vaccination process has provided benefits, the death toll remains frightening worldwide. Therefore, drugs and combined therapies that can be used against COVID-19 infection are still being investigated. Most of these antiviral medications are investigational drug candidates that are still in clinical trials. In this context, holistic and different approaches for the treatment of COVID-19, including prophylactic use of natural medicines, are under investigation and may offer potential treatment options due to the fact that this is still an unmet medical need of the world. Thus, inhibiting the increased glycolysis in COVID-19 infection with glycolysis inhibitors may be beneficial for patient survival. This short review highlights the potential benefits of glycolysis inhibition as well as controlling the elevated glucose levels in patients with COVID-19.

The Antiviral Effects of 2-Deoxy-D-glucose (2-DG), a Dual D-Glucose and D-Mannose Mimetic, against SARS-CoV-2 and Other Highly Pathogenic Viruses.

Viral infection almost invariably causes metabolic changes in the infected cell and several types of host cells that respond to the infection. Among metabolic changes, the most prominent is the upregulated glycolysis process as the main pathway of glucose utilization. Glycolysis activation is a common mechanism of cell adaptation to several viral infections, including noroviruses, rhinoviruses, influenza virus, Zika virus, cytomegalovirus, coronaviruses and others. Such metabolic changes provide potential targets for therapeutic approaches that could reduce the impact of infection. Glycolysis inhibitors, especially 2-deoxy-D-glucose (2-DG), have been intensively studied as antiviral agents. However, 2-DG's poor pharmacokinetic properties limit its wide clinical application. Herein, we discuss the potential of 2-DG and its novel analogs as potent promising antiviral drugs with special emphasis on targeted intracellular processes.

Elevated plasma level of the glycolysis byproduct methylglyoxal on admission is an independent biomarker of mortality in ICU COVID-19 patients.

Biomarkers to identify ICU COVID-19 patients at high risk for mortality are urgently needed for therapeutic care and management. Here we found plasma levels of the glycolysis byproduct methylglyoxal (MG) were 4.4-fold higher in ICU patients upon admission that later died (n = 33), and 1.7-fold higher in ICU patients that survived (n = 32),compared to uninfected controls (n = 30). The increased MG in patients that died correlated inversely with the levels of the MG-degrading enzyme glyoxalase-1 (r2 = - 0.50), and its co-factor glutathione (r2 = - 0.63), and positively with monocytes (r2 = 0.29). The inflammation markers, SSAO (r2 = 0.52), TNF-α (r2 = 0.41), IL-1ß (r2 = 0.25), CRP (r2 = 0.26) also correlated positively with MG. Logistic regression analysis provides evidence of a significant relationship between the elevated MG upon admission into ICU and death (P < 0.0001), with 42% of the death variability explained. From these data we conclude that elevated plasma MG on admission is a novel independent biomarker that predicts mortality in ICU COVID-19 patients.

Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I-Mediated Antiviral Responses in Primary Human Dendritic Cells.

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5' triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)-stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

TNFα-induced metabolic reprogramming drives an intrinsic anti-viral state.

Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of hexokinase 2 (HK2), the isoform predominantly expressed in muscle. Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα's ability to limit the replication of evolutionarily divergent viruses. TNFα was also found to modulate the metabolism of UDP-sugars, which are essential precursor substrates for glycosylation. Our data indicate that TNFα increases the concentration of UDP-glucose, as well as the glucose-derived labeling of UDP-glucose and UDP-N-acetyl-glucosamine in a glycolytically-dependent manner. Glycolysis was also necessary for the TNFα-mediated accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition attenuated TNFα's ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.

Haematopoietic ageing through the lens of single-cell technologies.

Human lifespan is now longer than ever and, as a result, modern society is getting older. Despite that, the detailed mechanisms behind the ageing process and its impact on various tissues and organs remain obscure. In general, changes in DNA, RNA and protein structure throughout life impair their function. Haematopoietic ageing refers to the age-related changes affecting a haematopoietic system. Aged blood cells display different functional aberrations depending on their cell type, which might lead to the development of haematologic disorders, including leukaemias, anaemia or declining immunity. In contrast to traditional bulk assays, which are not suitable to dissect cell-to-cell variation, single-cell-level analysis provides unprecedented insight into the dynamics of age-associated changes in blood. In this Review, we summarise recent studies that dissect haematopoietic ageing at the single-cell level. We discuss what cellular changes occur during haematopoietic ageing at the genomic, transcriptomic, epigenomic and metabolomic level, and provide an overview of the benefits of investigating those changes with single-cell precision. We conclude by considering the potential clinical applications of single-cell techniques in geriatric haematology, focusing on the impact on haematopoietic stem cell transplantation in the elderly and infection studies, including recent COVID-19 research.

Roles of Interleukin-6-mediated immunometabolic reprogramming in COVID-19 and other viral infection-associated diseases.

Interleukin-6 (IL-6) is a highly pleiotropic glycoprotein factor that can modulate innate and adaptive immunity as well as various aspects of metabolism, including glycolysis, fatty acid oxidation and oxidative phosphorylation. Recently, the expression and release of IL-6 is shown to be significantly increased in numerous diseases related to virus infection, and this increase is positively correlated with the disease severity. Immunity and metabolism are two highly integrated and interdependent systems, the balance between them plays a pivotal role in maintaining body homeostasis. IL-6-elicited inflammatory response is found to be closely associated with metabolic disorder in patients with viral infection. This brief review summarizes the regulatory role of IL-6 in immunometabolic reprogramming among seven viral infection-associated diseases.

A Comprehensive Biological and Synthetic Perspective on 2-Deoxy-d-Glucose (2-DG), A Sweet Molecule with Therapeutic and Diagnostic Potentials.

Glucose, the primary substrate for ATP synthesis, is catabolized during glycolysis to generate ATP and precursors for the synthesis of other vital biomolecules. Opportunistic viruses and cancer cells often hijack this metabolic machinery to obtain energy and components needed for their replication and proliferation. One way to halt such energy-dependent processes is by interfering with the glycolytic pathway. 2-Deoxy-d-glucose (2-DG) is a synthetic glucose analogue that can inhibit key enzymes in the glycolytic pathway. The efficacy of 2-DG has been reported across an array of diseases and disorders, thereby demonstrating its broad therapeutic potential. Recent approval of 2-DG in India as a therapeutic approach for the management of the COVID-19 pandemic has brought renewed attention to this molecule. The purpose of this perspective is to present updated therapeutic avenues as well as a variety of chemical synthetic strategies for this medically useful sugar derivative, 2-DG.

Glycolytic inhibitor 2-deoxy-d-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions.

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.

An airway organoid-based screen identifies a role for the HIF1α-glycolysis axis in SARS-CoV-2 infection.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.

Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets.

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.

Role of miR-2392 in driving SARS-CoV-2 infection.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provide an exciting avenue toward antiviral therapeutics. From patient transcriptomic data, we determined that a circulating miRNA, miR-2392, is directly involved with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia, as well as promoting many symptoms associated with coronavirus disease 2019 (COVID-19) infection. We demonstrate that miR-2392 is present in the blood and urine of patients positive for COVID-19 but is not present in patients negative for COVID-19. These findings indicate the potential for developing a minimally invasive COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we design a miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters, and may potentially inhibit a COVID-19 disease state in humans.

Inhibiting the reproduction of SARS-CoV-2 through perturbations in human lung cell metabolic network.

Viruses rely on their host for reproduction. Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction. Some of the catalysing enzymes of such reactions have demonstrated interactions with existing drugs, which can be used for experimental testing of the presented predictions using gene knockouts and RNA interference techniques. In summary, the developed computational approach offers a platform for rapid, experimentally testable generation of drug predictions against existing and emerging viruses based on their biomass requirements.

Genome-scale metabolic modelling of SARS-CoV-2 in cancer cells reveals an increased shift to glycolytic energy production.

Cancer is considered a high-risk condition for severe illness resulting from COVID-19. The interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and human metabolism is key to elucidating the risk posed by COVID-19 for cancer patients and identifying effective treatments, yet it is largely uncharacterised on a mechanistic level. We present a genome-scale map of short-term metabolic alterations triggered by SARS-CoV-2 infection of cancer cells. Through transcriptomic- and proteomic-informed genome-scale metabolic modelling, we characterise the role of RNA and fatty acid biosynthesis in conjunction with a rewiring in energy production pathways and enhanced cytokine secretion. These findings link together complementary aspects of viral invasion of cancer cells, while providing mechanistic insights that can inform the development of treatment strategies.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+ /NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a "shock and kill effect" decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.