The emerging roles of glutamine amidotransferases in metabolism and immune defense.

Glutamine amidotransferases (GATs) catalyze the synthesis of nucleotides, amino acids, glycoproteins and an enzyme cofactor, thus serving as key metabolic enzymes for cell proliferation. Carbamoyl-phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase (CAD) is a multifunctional enzyme of the GAT family and catalyzes the first three steps of the de novo pyrimidine synthesis. Following our findings that cellular GATs are involved in immune evasion during herpesvirus infection, we discovered that CAD reprograms cellular metabolism to fuel aerobic glycolysis and nucleotide synthesis via deamidating RelA. Deamidated RelA activates the expression of key glycolytic enzymes, rather than that of the inflammatory NF-κB-responsive genes. As such, cancer cells prime RelA for deamidation via up-regulating CAD activity or accumulating RelA mutations. Interestingly, the recently emerged SARS-CoV-2 also activates CAD to couple evasion of inflammatory response to activated nucleotide synthesis. A small molecule inhibitor of CAD depletes nucleotide supply and boosts antiviral inflammatory response, thus greatly reducing SARS-CoV-2 replication. Additionally, we also found that CTP synthase 1 (CTPS1) deamidates interferon (IFN) regulatory factor 3 (IRF3) to mute IFN induction. Our previous studies have implicated phosphoribosyl formylglycinamidine synthase (PFAS) and phosphoribosyl pyrophosphate amidotransferase (PPAT) in deamidating retinoic acid-inducible gene I (RIG-I) and evading dsRNA-induced innate immune defense in herpesvirus infection. Overall, these studies have uncovered an unconventional enzymatic activity of cellular GATs in metabolism and immune defense, offering a molecular link intimately coupling these fundamental biological processes.

The Interferon-inducible NAMPT acts as a protein phosphoribosylase to restrict viral infection.

As obligate intracellular pathogens, viruses often activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the salvage NAD+ synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses with unknown mechanism. Here we report that NAMPT restricts herpes simplex virus 1 (HSV-1) replication via phosphoribosyl-hydrolase activity toward key viral structural proteins, independent of NAD+ synthesis. Deep mining of enriched phosphopeptides of HSV-1-infected cells identified phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins. Indeed, NAMPT de-phosphoribosylates viral proteins in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Moreover, loss of NAMPT renders mice highly susceptible to HSV-1 infection. The work describes a hidden enzyme activity of a metabolic enzyme in viral infection and host defense, offering a system to interrogate roles of phosphoribosylation in metazoans.

Metabolic Enzymes in Viral Infection and Host Innate Immunity.

Metabolic enzymes are central players for cell metabolism and cell proliferation. These enzymes perform distinct functions in various cellular processes, such as cell metabolism and immune defense. Because viral infections inevitably trigger host immune activation, viruses have evolved diverse strategies to blunt or exploit the host immune response to enable viral replication. Meanwhile, viruses hijack key cellular metabolic enzymes to reprogram metabolism, which generates the necessary biomolecules for viral replication. An emerging theme arising from the metabolic studies of viral infection is that metabolic enzymes are key players of immune response and, conversely, immune components regulate cellular metabolism, revealing unexpected communication between these two fundamental processes that are otherwise disjointed. This review aims to summarize our present comprehension of the involvement of metabolic enzymes in viral infections and host immunity and to provide insights for potential antiviral therapy targeting metabolic enzymes.

SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

Interleukin-7 protects CD8+ T cells from adenosine-mediated immunosuppression.

The nucleoside adenosine accumulates extracellularly in solid tumors and inhibits CD8+ T cells by activating adenosine receptors. The cytokine interleukin-7 (IL-7), which is produced by various tissues and tumors, promotes the survival and maintenance of T cells. Adenosine and IL-7 signaling are being clinically targeted separately or in combination with other therapies for solid tumor indications. Here, we found that IL-7 signaling promoted the accumulation of tumor-associated CD8+ T cells, in part, by preventing adenosine-mediated immunosuppression. Inhibition of the transcription factor FoxO1 downstream of IL-7 receptor signaling was important for protecting CD8+ T cells from suppression by adenosine. These findings have implications for the development of new approaches for cancer immunotherapies that target the adenosine pathway.

Targeting CTP Synthetase 1 to Restore Interferon Induction and Impede Nucleotide Synthesis in SARS-CoV-2 Infection.

The newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.

Viral pseudoenzymes in infection and immunity.

Pseudoenzymes are proteins that are evolutionarily related to active enzymes, but lack relevant catalytic activity. As obligate intracellular pathogens, viruses complete their life cycle fully dependent on the cellular supplies of macromolecule and energy. Traditionally, studies of viral proteins sharing high homology with host counterparts reveal insightful mechanisms by which host signaling pathways are delicately regulated. Recent investigations into the action of cellular pseudoenzymes elucidate diverse molecular means how enzymes are differentially controlled under various physiological conditions, hinting to the potential that pathogens may exploit these regulatory modalities. To date, there have been three types of viral pseudoenzymes reported and our understanding concerning their mechanism of regulation is rudimentary at best. However, it is clear that viral pseudoenzymes are emerging with surprising functions in infection and immunity, and we are only at the beginning to understand this new group of enzyme regulators. In this review, we will summarize current knowledge in viral pseudoenzymes and provide a perspective for future research.

Deamidation Shunts RelA from Mediating Inflammation to Aerobic Glycolysis.

Cell proliferation and inflammation are two metabolically demanding biological processes. How these competing processes are selectively executed in the same cell remains unknown. Here, we report that the enzyme carbamoyl-phosphate synthetase, aspartyl transcarbamoylase, and dihydroorotase (CAD) deamidates the RelA subunit of NF-κB in cancer cells to promote aerobic glycolysis and fuel cell proliferation in tumorigenesis. This post-translational modification switches RelA function from mediating the expression of NF-κB-responsive genes to that of glycolytic enzymes, thus shunting the cell's inflammatory response to aerobic glycolysis. Further, we profiled diverse human cancer cell lines and found that high CAD expression and a subset of RELA mutations correlated with RelA deamidation. And by use of inhibitors of key glycolytic enzymes, we validated the pivotal role of RelA deamidation in tumorigenesis of cancer cell lines. This work illuminates a mechanism by which protein deamidation selectively specifies gene expression and consequent biological processes.

Adenosine Receptor Signaling Targets Both PKA and Epac Pathways to Polarize Dendritic Cells to a Suppressive Phenotype.

Extracellular adenosine accumulates in tumors and causes suppression of immune cells. Suppressive adenosine signaling is achieved through adenosine A2A and A2B receptors, which are Gs coupled, and their activation elevates cAMP levels. Gs-coupled GPCR signaling causes cAMP accumulation, which plays an anti-inflammatory role in immune cells. Protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are two intracellular receptors of cAMP. In this study we showed that adenosine receptor signaling polarizes activated murine dendritic cells (DCs) into a tumor-promoting suppressive phenotype. Adenosine receptor signaling activates cAMP pathway and upregulates the negative regulators of NF-κB but does not influence phosphorylation of immediate inflammatory signaling molecules downstream of TLR signaling. Pharmacologic activation of both PKA and Epac pathways by specific cAMP analogues phenocopied the effects of adenosine signaling on murine DCs, such as suppression of proinflammatory cytokines, elevation of anti-inflammatory IL-10, increased expression of regulators of NF-κB pathway, and finally suppression of T cell activation. Inhibition of effector cytokine, IL-12p40 production, and increased immunosuppressive IL-10 production by adenosine signaling is significantly reversed only when both PKA and Epac pathways were inhibited together. Adenosine signaling increased IL-10 secretion while decreasing IL-12p40 secretion in human monocyte-derived DCs. Stimulation of both PKA and Epac pathways also caused combinatorial effects in regulation of IL-12p40 secretion in human monocyte-derived DCs. Interestingly, PKA signaling alone caused similar increase in IL-10 secretion to that of adenosine signaling in human monocyte-derived DCs. Our data suggest adenosine/cAMP signaling targets both PKA/Epac pathways to fully differentiate DCs into a suppressive phenotype.