SARS-CoV-2 Protein Nsp9 Is Involved in Viral Evasion through Interactions with Innate Immune Pathways.

The suppression of the host's innate antiviral immune response by SARS-CoV-2, a contributing factor to the severity of disease, has been considerably studied in recent years. Many of these studies have focused on the actions of the structural proteins of the virus because of their accessibility to host immunological components. However, less is known about SARS-CoV-2 nonstructural and accessory proteins in relation to viral evasion. Herein, we study SARS-CoV-2 nonstructural proteins Orf3a, Orf6, and Nsp9 in a mimicked virus-infected state using poly(I:C), a synthetic analog of viral dsRNA, that elicits the antiviral immune response. Through genome-wide expression profiling, we determined that Orf3a, Orf6, and Nsp9 all modulate the host antiviral signaling transcriptome to varying extents, uniquely suppressing aspects of innate immune signaling. Our data suggest that SARS-CoV-2 Nsp9 hinders viral detection through suppression of RIG-I expression and antagonizes the interferon antiviral cascade by downregulating NF-kB and TBK1. Our data point to unique molecular mechanisms through which the different SARS-CoV-2 proteins suppress immune signaling and promote viral evasion. Nsp9 in particular acts on major elements of the host antiviral pathways to impair the antiviral immune response.

3-Oxidopyridinium Ions Are Versatile Bioorthogonal Dipoles for Use in Cycloadditions with Cyclooctynes.

3-oxidopyridinium ions are water stable and soluble heteroaromatic betaines that behave as latent dipoles and undergo a wide variety of cycloadditions. Research into the cycloaddition reactions of 3-oxidopyridiniums was spearheaded by Alan R. Katritzky and collaborators from the early 1970s until the late 1980s, but they have yet to be used for bioorthogonal applications. Herein we report that 3-oxidopyridiniums can readily react with 4-dibenzocyclooctynol (DIBO), a common bioorthogonal handle, in a [3+2] cycloaddition. The mechanism was investigated by altering the electronics of the reaction by changing the substituent on the 5 position of the pyridinium. Electron-donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.3×10-4 s-1 with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M-1 s-1 with 5-amino-N-methyl-3-oxidopyridinium. 3-oxidopyridiniums' appreciable cycloaddition rates and compatibility with bioorthogonally relevant environments give them the potential to be used in a variety of bioconjugation applications.

Mechanistic Analysis of Bioorthogonal Double Strain-Promoted Alkyne-Nitrone Cycloadditions Involving Dibenzocyclooctadiyne.

The reactions of nitrones with cyclooctadiynes were studied to establish the relative rates of sequential reactions and to determine the limits and scope of this bioorthogonal chemistry. We have established the second-order rate constants for the consecutive additions of a variety of nitrones onto diyne and studied the structure-activity relationships via Hammett plots. Results show that the addition of the second nitrone to the monointermediate occurs significantly faster than the first, with both reactions being faster than analogous reactions with azides. Computational chemistry supports these observations. The rate of second addition increases with electron-deficient nitrones, as demonstrated by a large rho value of 2.08, suggesting that the reaction rate can be controlled by nitrone selectivity. To further investigate the kinetic parameters of the reaction, dinitrone monomers containing cyclic and diaryl-nitrones were designed for use in oligomerization applications. Oligomerization was used as a probe to test the limits of the reactivity and attempt to isolate monocycloaddition products. The oligomer formed from a cyclic nitrone reacts faster, and detailed MALDI mass spectrometry analysis shows that monoaddition products exist only transiently and are not isolatable. These studies inform on the scope and limits of this chemistry in a variety of applications. We successfully demonstrated bacterial cell wall labeling using heterogeneous dual cycloadditions involving nitrone and azide dipoles, where the nitrone was the faster reacting partner on the bacterial cell surface.

microRNA-185 Inhibits SARS-CoV-2 Infection through the Modulation of the Host's Lipid Microenvironment.

With the emergence of the novel betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there has been an urgent need for the development of fast-acting antivirals, particularly in dealing with different variants of concern (VOC). SARS-CoV-2, like other RNA viruses, depends on host cell machinery to propagate and misregulate metabolic pathways to its advantage. Herein, we discovered that the immunometabolic microRNA-185 (miR-185) restricts SARS-CoV-2 propagation by affecting its entry and infectivity. The antiviral effects of miR-185 were studied in SARS-CoV-2 Spike protein pseudotyped virus, surrogate virus (HCoV-229E), as well as live SARS-CoV-2 virus in Huh7, A549, and Calu-3 cells. In each model, we consistently observed microRNA-induced reduction in lipid metabolism pathways-associated genes including SREBP2, SQLE, PPARG, AGPAT3, and SCARB1. Interestingly, we also observed changes in angiotensin-converting enzyme 2 (ACE2) levels, the entry receptor for SARS-CoV-2. Taken together, these data show that miR-185 significantly restricts host metabolic and other pathways that appear to be essential to SAR-CoV-2 replication and propagation. Overall, this study highlights an important link between non-coding RNAs, immunometabolic pathways, and viral infection. miR-185 mimics alone or in combination with other antiviral therapeutics represent possible future fast-acting antiviral strategies that are likely to be broadly antiviral against multiple variants as well as different virus types of potential pandemics.

An unnatural enzyme with endonuclease activity towards small non-coding RNAs.

Endonucleases are enzymes that cleave internal phosphodiester bonds within double-stranded DNA or RNA and are essential for biological functions. Herein, we use genetic code expansion to create an unnatural endonuclease that cleaves non-coding RNAs including short interfering RNA (siRNA) and microRNAs (miRNAs), a function that does not exist in nature. We introduce a metal-chelating unnatural amino acid, (2,2'-bipyridin-5-yl)alanine (BpyAla) to impart endonuclease activity to the viral suppressor of RNA silencing protein p19. Upon binding of copper, the mutant p19-T111BpyAla displays catalytic site-specific cleavage of siRNA and human miRNAs. Catalysis is confirmed using fluorescence polarization and fluorescence turn-on. Global miRNA profiling reveals that the engineered enzyme cleaves miRNAs in a human cell line. The therapeutic potential is demonstrated by targeting miR-122, a critical host factor for the hepatitis C virus (HCV). Unnatural endonuclease function is shown to deplete miR-122 levels with similar effects to an antagomir that reduces HCV levels therapeutically.

Optimized Bioorthogonal Non-canonical Amino Acid Tagging to Identify Serotype-Specific Biomarkers in Verotoxigenic Escherichia coli.

According to Canada's Food Report Card 2016, there are 4 million foodborne illnesses acquired each year in the nation alone. The leading causes of foodborne illness are pathogenic bacteria such as shigatoxigenic/verotoxigenic Escherichia coli (STEC/VTEC) and Listeria monocytogenes. Most current detection methods used to identify these bacterial pathogens are limited in their validity since they are not specific to detecting metabolically active organisms, potentially generating false-positive results from non-living or non-viable bacteria. Previously, our lab developed an optimized bioorthogonal non-canonical amino acid tagging (BONCAT) method which allows for the labeling of translationally active wild-type pathogenic bacteria. Incorporation of homopropargyl glycine (HPG) into the cellular surfaces of bacteria allows for protein tagging using the bioorthogonal alkyne handle to report on the presence of pathogenic bacteria. Here, we use proteomics to identify more than 400 proteins differentially detected by BONCAT between at least two of five different VTEC serotypes. These findings pave the way for future examination of these proteins as biomarkers in BONCAT-utilizing assays.

Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization.

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.

microRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection.

miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels.

miR-383 Regulates Hepatic Lipid Homeostasis and Response to Dengue Virus Infection.

Recently, microRNAs (miRNAs), as endogenous noncoding RNAs that inhibit mRNA translation, have been identified to broadly possess functional roles in regulating cellular signaling and metabolic processes due to their chemical and biological properties. In addition, they have emerged to be of critical importance in modulating host-virus interactions, especially for RNA viruses. Herein, we discovered that miR-383-5p targets certain lipid and cholesterol biosynthetic pathways and restricts Dengue virus (DENV) infection in hepatic cells. Global transcriptomics analysis of Huh7 human hepatoma cells overexpressing miR-383-5p revealed enrichment of lipid and cholesterol metabolic processes. Bioinformatics analysis of genes repressed in miR-383-5p overexpressing cells divulged the repression of a key target PLA2G4A, a pro-viral host factor essential for the production of infectious DENV particles. Our study demonstrated the effectiveness of miRNA mimics as tools to study cellular signaling pathways that contribute to viral pathogenesis. Overall, our study identifies miR-383-5p as an interesting host factor during DENV propagation and highlights a potential therapeutic role in the regulation of hepatic lipid metabolism and an antiviral response to DENV.

Bioorthogonal chemistry.

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide-alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of 'click chemistry' - high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody-drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

Armeniaspirols inhibit the AAA+ proteases ClpXP and ClpYQ leading to cell division arrest in Gram-positive bacteria.

Multi-drug-resistant bacteria present an urgent threat to modern medicine, creating a desperate need for antibiotics with new modes of action. As natural products remain an unsurpassed source for clinically viable antibiotic compounds, we investigate the mechanism of action of armeniaspirol. The armeniaspirols are a structurally unique class of Gram-positive antibiotic discovered from Streptomyces armeniacus for which resistance cannot be readily obtained. We show that armeniaspirol inhibits the ATP-dependent proteases ClpXP and ClpYQ in vitro and in the model Gram-positive Bacillus subtilis. This inhibition dysregulates the divisome and elongasome supported by an upregulation of key proteins FtsZ, DivIVA, and MreB inducing cell division arrest. The inhibition of ClpXP and ClpYQ to dysregulate cell division represents a unique antibiotic mechanism of action and armeniaspirol is the only known natural product inhibitor of the coveted anti-virulence target ClpP. Thus, armeniaspirol possesses a promising lead scaffold for antibiotic development with unique pharmacology.

Reactivity of N-acyl hydrazone probes with the mammalian proteome.

Small molecule probes with distinct reactivities are useful tools for the identification and characterization of protein modifications and function. Herein, we show that hydrazone probes with an N-carbamate structural motif react differently from N-carbamates within the human proteome. Mass spectrometry analysis of probe-treated mammalian cell lysates identified several proteins that were covalently modified by the hydrazone probes, including the cytidine deaminase APOBEC3A. We used this enzyme as a model to explore the reactivity of the probes with amino acid residues using LC-MS/MS. Both reactive serine and cysteine residues outside of the enzyme active site were covalently modified. A 1-napthol leaving group provided the most extensive reactivity. These results confirm a unique chemotype for hydrazone probes which can be further optimized to target distinct targets of the human proteome.

MicroRNA Mimics or Inhibitors as Antiviral Therapeutic Approaches Against COVID-19.

Coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 (COVID-19) pandemic, present a significant threat to human health by inflicting a wide variety of health complications and even death. While conventional therapeutics often involve administering small molecules to fight viral infections, small non-coding RNA sequences, known as microRNAs (miRNAs/miR-), may present a novel antiviral strategy. We can take advantage of their ability to modulate host-virus interactions through mediating RNA degradation or translational inhibition. Investigations into miRNA and SARS-CoV-2 interactions can reveal novel therapeutic approaches against this virus. The viral genomes of SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) were searched using the Nucleotide Basic Local Alignment Search Tool (BLASTn) for highly similar sequences, to identify potential binding sites for miRNAs hypothesized to play a role in SARS-CoV-2 infection. miRNAs that target angiotensin-converting enzyme 2 (ACE2), the receptor used by SARS-CoV-2 and SARS-CoV for host cell entry, were also predicted. Several relevant miRNAs were identified, and their potential roles in regulating SARS-CoV-2 infections were further assessed. Current treatment options for SARS-CoV-2 are limited and have not generated sufficient evidence on safety and efficacy for treating COVID-19. Therefore, by investigating the interactions between miRNAs and SARS-CoV-2, miRNA-based antiviral therapies, including miRNA mimics and inhibitors, may be developed as an alternative strategy to fight COVID-19.

Profiling of MicroRNA Targets Using Activity-Based Protein Profiling: Linking Enzyme Activity to MicroRNA-185 Function.

MicroRNAs (miRNAs) act as cellular signal transducers through repression of protein translation. Elucidating targets using bioinformatics and traditional quantitation methods is often insufficient to uncover global miRNA function. Herein, alteration of protein function caused by miRNA-185 (miR-185), an immunometabolic miRNA, was determined using activity-based protein profiling, transcriptomics, and lipidomics. Fluorophosphonate-based activity-based protein profiling of miR-185-induced changes to human liver cells revealed that exclusively metabolic serine hydrolase enzymes were regulated in activity, some with roles in lipid and endocannabinoid metabolism. Lipidomic analysis linked enzymatic changes to levels of cellular lipid species, such as components of very-low-density lipoprotein particles. Additionally, inhibition of one miR-185 target, monoglyceride lipase, led to decreased hepatitis C virus levels in an infectious model. Overall, the approaches used here were able to identify key functional changes in serine hydrolases caused by miR-185 that are targetable pharmacologically, such that a small molecule inhibitor can recapitulate the miRNA phenotype.

Bioorthogonal Reactions Utilizing Nitrones as Versatile Dipoles in Cycloaddition Reactions.

Bioorthogonal chemical reactions have emerged as convenient and rapid methods for incorporating unnatural functionality into living systems. Different prototype reactions have been optimized for use in biological settings. Optimization of 3 + 2 dipolar cycloadditions involving nitrones has resulted in highly efficient reaction conditions for bioorthogonal chemistry. Through substitution at the nitrone carbon or nitrogen atom, stereoelectronic tuning of the reactivity of the dipole has assisted in optimizing reactivity. Nitrones have been shown to react rapidly with cyclooctynes with bimolecular rate constants approaching k2 = 102 M-1 s-1, which are among the fastest bioorthogonal reactions reported (McKay et al. Org. Biomol. Chem. 2012, 10, 3066-3070). Nitrones have also been shown to react with trans-cyclooctenes (TCO) in strain-promoted TCO-nitrone cycloadditions reactions. Copper catalyzed reactions involving alkynes and nitrones have also been optimized for applications in biology. This review provides a comprehensive accounting of the different bioorthogonal reactions that have been developed using nitrones as versatile reactants, and provides some recent examples of applications for probing biological systems.

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism.

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.

Optimized aqueous Kinugasa reactions for bioorthogonal chemistry applications.

Kinugasa reactions hold potential for bioorthogonal chemistry in that the reagents can be biocompatible. Unlike other bioorthogonal reaction products, ß-lactams are potentially reactive, which can be useful for synthesizing new biomaterials. A limiting factor for applications consists of slow reaction rates. Herein, we report an optimized aqueous copper(i)-catalyzed alkyne-nitrone cycloaddition involving rearrangement (CuANCR) with rate accelerations made possible by the use of surfactant micelles. We have investigated the factors that accelerate the aqueous CuANCR reaction and demonstrate enhanced modification of a model membrane-associated peptide. We discovered that lipids/surfactants and alkyne structure have a significant impact on the reaction rate, with biological lipids and electron-poor alkynes showing greater reactivity. These new findings have implications for the use of CuANCR for modifying integral membrane proteins as well as live cell labelling and other bioorthogonal applications.