Human ACE2 expression, a major tropism determinant for SARS-CoV-2, is regulated by upstream and intragenic elements.

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19.

Strategic Fluorination to Achieve a Potent, Selective, Metabolically Stable, and Orally Bioavailable Inhibitor of CSNK2.

The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against ß-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition.

Novel modulators of p53-signaling encoded by unknown genes of emerging viruses.

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs.

Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.

Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner.

Inhalational exposure to particulate matter (PM) derived from natural or anthropogenic sources alters gene expression in the airways and increases susceptibility to respiratory viral infection. Woodsmoke-derived ambient PM from wildfire events during 2020 was associated with higher COVID-19 case rates in the western United States. We hypothesized that exposure to suspensions of woodsmoke particles (WSPs) or diesel exhaust particles (DEPs) prior to SARS-CoV-2 infection would alter host immune gene expression at the transcript level. Primary human nasal epithelial cells (hNECs) from both sexes were exposed to WSPs or DEPs (22 µg/cm2) for 2 h, followed by infection with SARS-CoV-2 at a multiplicity of infection of 0.5. Forty-six genes related to SARS-CoV-2 entry and host response were assessed. Particle exposure alone minimally affected gene expression, whereas SARS-CoV-2 infection alone induced a robust transcriptional response in hNECs, upregulating type I and III interferons, interferon-stimulated genes, and chemokines by 72 h postinfection (p.i.). This upregulation was higher overall in cells from male donors. However, exposure to WSPs prior to infection dampened expression of antiviral, interferon, and chemokine mRNAs. Sex stratification of these results revealed that WSP exposure downregulated gene expression in cells from females more so than males. We next hypothesized that hNECs exposed to particles would have increased apical viral loads compared with unexposed cells. Although apical viral load was correlated to expression of host response genes, viral titer did not differ between groups. These data indicate that WSPs alter epithelial immune responses in a sex-dependent manner, potentially suppressing host defense to SARS-CoV-2 infection.

Identification of Quinolinones as Antivirals against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Using SHAPE-MaP To Model RNA Secondary Structure and Identify 3'UTR Variation in Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.

Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.

BACKGROUND: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. RESULTS: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. CONCLUSION: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

More than an Amide Bioisostere: Discovery of 1,2,4-Triazole-containing Pyrazolo[1,5-a]pyrimidine Host CSNK2 Inhibitors for Combatting ß-Coronavirus Replication.

The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against ß-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.

Host Genetic Variation Impacts SARS-CoV-2 Vaccination Response in the Diversity Outbred Mouse Population.

The COVID-19 pandemic led to the rapid and worldwide development of highly effective vaccines against SARS-CoV-2. However, there is significant individual-to-individual variation in vaccine efficacy due to factors including viral variants, host age, immune status, environmental and host genetic factors. Understanding those determinants driving this variation may inform the development of more broadly protective vaccine strategies. While host genetic factors are known to impact vaccine efficacy for respiratory pathogens such as influenza and tuberculosis, the impact of host genetic variation on vaccine efficacy against COVID-19 is not well understood. To model the impact of host genetic variation on SARS-CoV-2 vaccine efficacy, while controlling for the impact of non-genetic factors, we used the Diversity Outbred (DO) mouse model. We found that DO mice immunized against SARS-CoV-2 exhibited high levels of variation in vaccine-induced neutralizing antibody responses. While the majority of the vaccinated mice were protected from virus-induced disease, similar to human populations, we observed vaccine breakthrough in a subset of mice. Importantly, we found that this variation in neutralizing antibody, virus-induced disease, and viral titer is heritable, indicating that the DO serves as a useful model system for studying the contribution of genetic variation of both vaccines and disease outcomes.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

A single-dose intranasal live-attenuated codon deoptimized vaccine provides broad protection against SARS-CoV-2 and its variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) continues its significant health and economic impact globally. Despite the success of spike-protein vaccines in preventing severe disease, long-lasting protection against emerging variants and the prevention of breakthrough infections and transmission remain elusive. We generate an intranasal live-attenuated SARS-CoV-2 vaccine, CDO-7N-1, using codon deoptimization. CDO-7N-1 shows highly attenuated replication and minimal or no lung pathology in vivo over multiple passages. It induces robust mucosal and systemic neutralizing antibody and T-cell subset responses, in mice (female K18-hACE2 and male HFH4-hACE2 mice), hamsters, and macaques triggered by a single immunization. Mice and hamsters vaccinated with CDO-7N-1 are protected from challenge with wild-type (WT) SARS-CoV-2 and other variants of concern. Serum from vaccinated animals neutralizes WT SARS-CoV-2, variants of concern (beta and delta), variants of interest (omicron XBB.1.5) and SARS-CoV-1. Antibody responses are sustained and enhanced by repeated immunization or infection with WT SARS-CoV-2. Immunity against all SARS-CoV-2 proteins by CDO-7N-1 should improve efficacy against future SARS-CoV-2 variants.

PanG, a new ketopantoate reductase involved in pantothenate synthesis.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2.

Elevated levels of cytokines IL-1ß and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1ß released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1ß release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1ß release. After release, IL-1ß stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1ß secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1ß and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Adjuvant-dependent effects on the safety and efficacy of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Inactivated whole virus SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide (Alum) are among the most widely used COVID-19 vaccines globally and have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous virus infection in healthy recipients, the emergence of novel SARS-CoV-2 variants and the presence of large zoonotic reservoirs provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes including vaccine-associated enhanced respiratory disease (VAERD). To evaluate this possibility, we tested the performance of an inactivated SARS-CoV-2 vaccine (iCoV2) in combination with Alum against either homologous or heterologous coronavirus challenge in a mouse model of coronavirus-induced pulmonary disease. Consistent with human results, iCoV2 + Alum protected against homologous challenge. However, challenge with a heterologous SARS-related coronavirus, Rs-SHC014-CoV (SHC014), up to at least 10 months post-vaccination, resulted in VAERD in iCoV2 + Alum-vaccinated animals, characterized by pulmonary eosinophilic infiltrates, enhanced pulmonary pathology, delayed viral clearance, and decreased pulmonary function. In contrast, vaccination with iCoV2 in combination with an alternative adjuvant (RIBI) did not induce VAERD and promoted enhanced SHC014 clearance. Further characterization of iCoV2 + Alum-induced immunity suggested that CD4+ T cells were a major driver of VAERD, and these responses were partially reversed by re-boosting with recombinant Spike protein + RIBI adjuvant. These results highlight potential risks associated with vaccine breakthrough in recipients of Alum-adjuvanted inactivated vaccines and provide important insights into factors affecting both the safety and efficacy of coronavirus vaccines in the face of heterologous virus infections.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Francisella tularensis RipA protein topology and identification of functional domains.

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1ß (IL-1ß) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of ß-Coronaviruses.

From a designed library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (17) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine (30) was characterized as a negative control that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled broadly. Chemical probe 17 disrupts multiple phases of the lifecycle of ß-coronaviruses: viral replication and viral entry. The diverse antiviral roles of PIKfyve have not been previously probed comprehensively in a single study or using the same compound set. Our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has a non-overlapping kinase off-target profile with known PIKfyve inhibitors. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology.