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Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways.
Copyright © 2015 Elsevier Inc. All rights reserved.
UNLABELLED - Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development.
IMPORTANCE - Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Erythropoietin (EPO) is critical for red blood cell production and is also an effective neuroprotective agent. However, it may contribute to pathological angiogenesis. Here we investigate the angiogenic potential of EPO and a mutant form with attenuated erythropoietic activity, EPO-R76E, on primary human retinal microvascular endothelial cells (HRMECs) and in the adult retina. Assays of death, proliferation and tube formation were performed on HRMECs exposed to EPO, EPO-R76E or media alone. Postnatal day-9 wild-type mice were injected intramuscularly with adeno-associated virus vectors expressing either enhanced green fluorescent protein or EpoR76E. At 3 months, levels of EPO-R76E in the eye were quantified, and the health of the retinal vasculature was assessed by fluorescein angiography and isolectin immunolabeling. Immunohistochemistry, histology and electroretinogram (ERG) assessments were performed as measures of retinal health. Neither EPO nor EPO-R76E induced proliferation or tube formation in HRMECs under the conditions used. EPO-R76E decreased HRMEC death in a dose-dependent manner. Long-term systemic gene delivery of EPO-R76E was safe in terms of retinal vasculature, histology and the ERG in vivo. Our results show that EPO-R76E can block HRMEC death, consistent with its role in erythropoiesis and neuroprotection. In addition, long-term gene delivery of EPO-R76E is safe in the adult retina.
The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS.
© 2014 Society for Reproduction and Fertility.
Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. (13)C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive (13)C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.
Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
BACKGROUND - New drugs are routinely screened for IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway.
METHODS AND RESULTS - Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in INa-L. Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell-derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A, encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr-blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, d-sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil).
CONCLUSIONS - Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.
© 2014 American Heart Association, Inc.
Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.
© 2014 Society for Laboratory Automation and Screening.
UNLABELLED - Chikungunya virus (CHIKV) is a reemerging arbovirus responsible for outbreaks of infection throughout Asia and Africa, causing an acute illness characterized by fever, rash, and polyarthralgia. Although CHIKV infects a broad range of host cells, little is known about how CHIKV binds and gains access to the target cell interior. In this study, we tested whether glycosaminoglycan (GAG) binding is required for efficient CHIKV replication using CHIKV vaccine strain 181/25 and clinical isolate SL15649. Preincubation of strain 181/25, but not SL15649, with soluble GAGs resulted in dose-dependent inhibition of infection. While parental Chinese hamster ovary (CHO) cells are permissive for both strains, neither strain efficiently bound to or infected mutant CHO cells devoid of GAG expression. Although GAGs appear to be required for efficient binding of both strains, they exhibit differential requirements for GAGs, as SL15649 readily infected cells that express excess chondroitin sulfate but that are devoid of heparan sulfate, whereas 181/25 did not. We generated a panel of 181/25 and SL15649 variants containing reciprocal amino acid substitutions at positions 82 and 318 in the E2 glycoprotein. Reciprocal exchange at residue 82 resulted in a phenotype switch; Gly(82) results in efficient infection of mutant CHO cells but a decrease in heparin binding, whereas Arg(82) results in reduced infectivity of mutant cells and an increase in heparin binding. These results suggest that E2 residue 82 is a primary determinant of GAG utilization, which likely mediates attenuation of vaccine strain 181/25.
IMPORTANCE - Chikungunya virus (CHIKV) infection causes a debilitating rheumatic disease that can persist for months to years, and yet there are no licensed vaccines or antiviral therapies. Like other alphaviruses, CHIKV displays broad tissue tropism, which is thought to be influenced by virus-receptor interactions. In this study, we determined that cell-surface glycosaminoglycans are utilized by both a vaccine strain and a clinical isolate of CHIKV to mediate virus binding. We also identified an amino acid polymorphism in the viral E2 attachment protein that influences utilization of glycosaminoglycans. These data enhance an understanding of the viral and host determinants of CHIKV cell entry, which may foster development of new antivirals that act by blocking this key step in viral infection.
A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1-M4 IC50 > 30 μM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.