scholarly journals Entry by multiple picornaviruses is dependent on a pathway that includes TNK2, WASL and NCK1

2019 ◽  
Author(s):  
Hongbing Jiang ◽  
Christian Leung ◽  
Stephen Tahan ◽  
David Wang
Keyword(s):  
Encephalomyocarditis Virus ◽  
Epistasis Analysis ◽  
Human Genes ◽  
Comprehensive Knowledge ◽  
Chemical Inhibitors ◽  
Genetic Epistasis ◽  
Picornavirus Infection ◽  
Enterovirus D68 ◽  
Nucleation Activity ◽  
Viral Lifecycle

AbstractComprehensive knowledge of the host factors required for picornavirus infection would facilitate antiviral development. Here we demonstrate roles for three human genes, TNK2, WASL, and NCK1, in infection by multiple picornaviruses. CRISPR deletion of TNK2, WASL or NCK1 reduced encephalomyocarditis virus (EMCV), coxsackievirus B3 (CVB3), poliovirus and enterovirus D68 infection, and chemical inhibitors of TNK2 and WASL decreased EMCV infection. Reduced EMCV lethality was observed in mice lacking TNK2. TNK2, WASL and NCK1 were important in early stages of the viral lifecycle, and genetic epistasis analysis demonstrated that the three genes function in a common pathway. Mechanistically, reduced internalization of EMCV was observed in TNK2 deficient cells demonstrating that TNK2 functions in EMCV entry. Domain analysis of WASL demonstrated that its actin nucleation activity was necessary to facilitate viral infection. Together, these data support a model wherein TNK2, WASL, and NCK1 comprise a pathway critical for multiple picornaviruses.

scholarly journals Entry by multiple picornaviruses is dependent on a pathway that includes TNK2, WASL, and NCK1

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Hongbing Jiang ◽  
Christian Leung ◽  
Stephen Tahan ◽  
David Wang
Keyword(s):  
Encephalomyocarditis Virus ◽  
Epistasis Analysis ◽  
Human Genes ◽  
Comprehensive Knowledge ◽  
Chemical Inhibitors ◽  
Genetic Epistasis ◽  
Picornavirus Infection ◽  
Enterovirus D68 ◽  
Nucleation Activity ◽  
Viral Lifecycle

Comprehensive knowledge of the host factors required for picornavirus infection would facilitate antiviral development. Here we demonstrate roles for three human genes, TNK2, WASL, and NCK1, in infection by multiple picornaviruses. CRISPR deletion of TNK2, WASL, or NCK1 reduced encephalomyocarditis virus (EMCV), coxsackievirus B3 (CVB3), poliovirus and enterovirus D68 infection, and chemical inhibitors of TNK2 and WASL decreased EMCV infection. Reduced EMCV lethality was observed in mice lacking TNK2. TNK2, WASL, and NCK1 were important in early stages of the viral lifecycle, and genetic epistasis analysis demonstrated that the three genes function in a common pathway. Mechanistically, reduced internalization of EMCV was observed in TNK2 deficient cells demonstrating that TNK2 functions in EMCV entry. Domain analysis of WASL demonstrated that its actin nucleation activity was necessary to facilitate viral infection. Together, these data support a model wherein TNK2, WASL, and NCK1 comprise a pathway important for multiple picornaviruses.

scholarly journals Optimizing Dietary Restriction for Genetic Epistasis Analysis and Gene Discovery in C. elegans

PLoS ONE ◽  
2009 ◽  
Vol 4 (2) ◽  
pp. e4535 ◽  
Author(s):  
William Mair ◽  
Siler H. Panowski ◽  
Reuben J. Shaw ◽  
Andrew Dillin
Keyword(s):  
Dietary Restriction ◽  
Gene Discovery ◽  
C Elegans ◽  
Epistasis Analysis ◽  
Genetic Epistasis

Analysis of a Mutant Exhibiting Conditional Sorting to Dense Core Secretory Granules in Tetrahymena thermophila

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1605-1616
Author(s):  
Grant R Bowman ◽  
Aaron P Turkewitz
Keyword(s):  
De Novo ◽  
Tetrahymena Thermophila ◽  
Previous Analysis ◽  
Secretory Granules ◽  
Dense Core ◽  
Secretory Proteins ◽  
Epistasis Analysis ◽  
Complete Study ◽  
Genetic Epistasis ◽  
Dense Core Granules

Abstract The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.

scholarly journals Role of RIG-I-like receptors in innate immune sensing of Coxsackievirus B3 and encephalomyocarditis virus in murine macrophages and fibroblasts

2018 ◽  
Author(s):  
Esther Francisco ◽  
Mehul Suthar ◽  
Michael Gale ◽  
Amy B. Rosenfeld ◽  
Vincent R. Racaniello
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Coxsackievirus B3 ◽  
Innate Immune ◽  
Encephalomyocarditis Virus ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Murine Macrophages ◽  
Picornavirus Infection

AbstractViral infections are sensed by pattern recognition receptors that trigger an innate immune response through the expression of interferons (IFNs) and other cytokines. Most RNA viruses are sensed by the RIG-I like receptors (RLR)s. The contributions of these receptors to sensing viruses of thePicornaviridaefamily were investigated. Encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3), picornaviruses of theCardiovirusandEnterovirusgenera, are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFNβ synthesis is reduced in the absence of each sensor, while IFNα production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate that innate sensing of similar viruses by RLRs depends upon the cell type.ImportanceEnteroviruses such as Coxsackieviruses are the most common human respiratory pathogens. The host’s innate immune response, in particular that modulated by the production of type I and III interferons, is thought to restrict picornavirus infection. Two cytoplasmic proteins, MDA5 and RIG-I, are critical for initiating the early innate immune response against these viruses. Mutations within MDA5 encoding gene have been associated with the development of severe enterovirus associated respiratory illnesses in healthy children. To further understand how the innate immune response dependent upon MDA5 and Rig-I is initiated during picornavirus infection, macrophages from mice lacking MDA5 or RIG-I were infected with Coxsackievirus B3 (CVB3) and a related animal virus. RIG-I is essential for type I IFN production during CVB3 infection; when MDA5 is present, viral titers are reduced by an IFN-independent pathway. These observations demonstrate that innate sensing of viruses by MDA5 and RIG-I depends upon the cell type.

scholarly journals miR-146a–Traf6 regulatory axis controls autoimmunity and myelopoiesis, but is dispensable for hematopoietic stem cell homeostasis and tumor suppression

2017 ◽  
Vol 114 (34) ◽  
pp. E7140-E7149 ◽  
Author(s):  
Nathaniel Magilnick ◽  
Estefany Y. Reyes ◽  
Wei-Le Wang ◽  
Steven L. Vonderfecht ◽  
Jin Gohda ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Bone Marrow Failure ◽  
Biological Processes ◽  
Hematopoietic Stem ◽  
Epistasis Analysis ◽  
Signaling Axis ◽  
Genetic Epistasis ◽  
Molecular Brake

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a–mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a–Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a−/− mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a−/− phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a−/− mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.

scholarly journals Caveolin-1 Mediated Endocytic Pathway is Involved in Encephalomyocarditis Virus Replication in BHK-21 Cells

2020 ◽  
Author(s):  
Qiongyi Li ◽  
Yang Liu ◽  
Shujuan Xu ◽  
Kexue Zhao ◽  
Ying Ling ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Early Stage ◽  
Encephalomyocarditis Virus ◽  
Endocytic Pathway ◽  
Virus Infectivity ◽  
Key Factors ◽  
Caveolin 1 ◽  
Chemical Inhibitors ◽  
Microscopy Analysis

Abstract Background: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. Methods: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. Results: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. Conclusions: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

scholarly journals Nodal Modulator is required to sustain endoplasmic reticulum morphology

2021 ◽  
Author(s):  
Catherine Amaya ◽  
Christopher JF Cameron ◽  
Swapnil C. Devarkar ◽  
Mark B. Gerstein ◽  
Yong Xiong ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Integrity ◽  
Transmembrane Protein ◽  
Type I ◽  
Tail Domain ◽  
Epistasis Analysis ◽  
Proteomics Approach ◽  
Genetic Epistasis ◽  
String Structure

AbstractNodal Modulator (NOMO) is a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We identified NOMO1 in a proteomics approach aimed at the identification of proteins that support the structural integrity of the endoplasmic reticulum (ER). Overexpression of NOMO1 imposes a sheet morphology on the ER, while depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network. These structures are positive for the autophagy marker LAMP1, and LC3 is profoundly upregulated upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a dimeric state that is mediated by the cytosolic tail domain, with each monomer featuring a “beads on a string” structure likely representing bacterial Ig-like folds. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.

scholarly journals The NCA-1 and NCA-2 ion channels function downstream of Gq and Rho to regulate locomotion in C. elegans

2016 ◽  
Author(s):  
Irini Topalidou ◽  
Pin-An Chen ◽  
Kirsten Cooper ◽  
Shigeki Watanabe ◽  
Erik M. Jorgensen ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
C Elegans ◽  
Activating Mutations ◽  
Epistasis Analysis ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Forward Genetic Screens ◽  
Genetic Epistasis

AbstractThe heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans. Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans. Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

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