scholarly journals The Leader Protein of Theiler's Virus Prevents the Activation of PKR

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Teresa Cesaro ◽  
Thomas Michiels
Keyword(s):  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Granule Formation ◽  
Double Stranded Rna ◽  
Eif2α Phosphorylation ◽  
Leader Protein ◽  
Infected Cells ◽  
L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

scholarly journals Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells

2009 ◽  
Vol 84 (7) ◽  
pp. 3654-3665 ◽  
Author(s):  
Joanna Piotrowska ◽  
Spencer J. Hansen ◽  
Nogi Park ◽  
Katarzyna Jamka ◽  
Peter Sarnow ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Viral Infections ◽  
De Novo ◽  
Stress Granules ◽  
Stress Granule ◽  
Initiation Factor ◽  
Granule Formation ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACT Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.

scholarly journals Regulation of Stress Granule Formation by Inflammation, Vascular Injury, and Atherosclerosis

2019 ◽  
Vol 39 (10) ◽  
pp. 2014-2027 ◽  
Author(s):  
Allison B. Herman ◽  
Milessa Silva Afonso ◽  
Sheri E. Kelemen ◽  
Mitali Ray ◽  
Christine N. Vrakas ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Muscle Cells ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Granule Formation ◽  
Altered Expression

Objective: Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR − /− mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow–derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. Conclusions: These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.

scholarly journals Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

2014 ◽  
Vol 211 (2) ◽  
pp. 263-280 ◽  
Author(s):  
Hirofumi Sawada ◽  
Toshie Saito ◽  
Nils P. Nickel ◽  
Tero-Pekka Alastalo ◽  
Jason P. Glotzbach ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Mrna Translation ◽  
Inflammatory Cells ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Granule Formation ◽  
Gm Csf ◽  
Protein Receptor

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34–PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)–positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

scholarly journals Essential Role of Enterovirus 2A Protease in Counteracting Stress Granule Formation and the Induction of Type I Interferon

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Linda J. Visser ◽  
Martijn A. Langereis ◽  
Huib H. Rabouw ◽  
Maryam Wahedi ◽  
Elke M. Muntjewerff ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Cellular Responses ◽  
Encephalomyocarditis Virus ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Type I ◽  
Granule Formation ◽  
Α Subunit ◽  
Viral Proteases

ABSTRACTMost viruses have acquired mechanisms to suppress antiviral alpha/beta interferon (IFN-α/β) and stress responses. Enteroviruses (EVs) actively counteract the induction of IFN-α/β gene transcription and stress granule (SG) formation, which are increasingly implicated as a platform for antiviral signaling, but the underlying mechanisms remain poorly understood. Both viral proteases (2Aproand 3Cpro) have been implicated in the suppression of these responses, but these conclusions predominantly rely on ectopic overexpression of viral proteases or addition of purified viral proteases to cell lysates. Here, we present a detailed and comprehensive comparison of the effect of individual enterovirus proteases on the formation of SGs and the induction of IFN-α/β gene expression in infected cells for representative members of the enterovirus species EV-A to EV-D. First, we show that SG formation and IFN-β induction are suppressed in cells infected with EV-A71, coxsackie B3 virus (CV-B3), CV-A21, and EV-D68. By introducing genes encoding CV-B3 proteases in a recombinant encephalomyocarditis virus (EMCV) that was designed to efficiently activate antiviral responses, we show that CV-B3 2Apro, but not 3Cpro, is the major antagonist that counters SG formation and IFN-β gene transcription and that 2Apro’s proteolytic activity is essential for both functions. 2Aproefficiently suppressed SG formation despite protein kinase R (PKR) activation and α subunit of eukaryotic translation initiation factor 2 phosphorylation, suggesting that 2Aproantagonizes SG assembly or promotes its disassembly. Finally, we show that the ability to suppress SG formation and IFN-β gene transcription is conserved in the 2Aproof EV-A71, CV-A21, and EV-D68. Collectively, our results indicate that enterovirus 2Aproplays a key role in inhibiting innate antiviral cellular responses.IMPORTANCEEnteroviruses are important pathogens that can cause a variety of diseases in humans, including aseptic meningitis, myocarditis, hand-foot-and-mouth disease, conjunctivitis, and acute flaccid paralysis. Like many other viruses, enteroviruses must counteract antiviral cellular responses to establish an infection. It has been suggested that enterovirus proteases cleave cellular factors to perturb antiviral pathways, but the exact contribution of viral proteases 2Aproand 3Cproremains elusive. Here, we show that 2Apro, but not 3Cpro, of all four human EV species (EV-A to EV-D) inhibits SG formation and IFN-β gene transcription. Our observations suggest that enterovirus 2Aprohas a conserved function in counteracting antiviral host responses and thereby is the main enterovirus “security protein.” Understanding the molecular mechanisms of enterovirus immune evasion strategies may help to develop countermeasures to control infections with these viruses.

scholarly journals Inhibition of the Ubiquitin-Proteasome System Induces Stress Granule Formation

2007 ◽  
Vol 18 (7) ◽  
pp. 2603-2618 ◽  
Author(s):  
Rachid Mazroui ◽  
Sergio Di Marco ◽  
Randal J. Kaufman ◽  
Imed-Eddine Gallouzi
Keyword(s):  
Mrna Decay ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Early Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Granule Formation ◽  
Eif2α Phosphorylation

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2α by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2α phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21cip1 mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.

scholarly journals PRMT7 methylates eukaryotic translation initiation factor 2α and regulates its role in stress granule formation

2019 ◽  
Vol 30 (6) ◽  
pp. 778-793 ◽  
Author(s):  
Nasim Haghandish ◽  
R. Mitchell Baldwin ◽  
Alan Morettin ◽  
Haben Tesfu Dawit ◽  
Hemanta Adhikary ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Single Type ◽  
Granule Formation ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Arginine Residues ◽  
Eukaryotic Translation Initiation

Protein arginine methyltransferases (PRMTs) are a family of enzymes that modify proteins by methylating the guanidino nitrogen atoms of arginine residues to regulate cellular processes such as chromatin remodeling, pre-mRNA splicing, and signal transduction. PRMT7 is the single type III PRMT solely capable of arginine monomethylation. To date, other than histone proteins, there are very few identified substrates of PRMT7. We therefore performed quantitative mass spectrometry experiments to identify PRMT7’s interactome and potential substrates to better characterize the enzyme’s biological function(s) in cells. These experiments revealed that PRMT7 interacts with and can methylate eukaryotic translation initiation factor 2 alpha (eIF2α), in vitro and in breast cancer cells. Furthermore, we uncovered a potential regulatory interplay between eIF2α arginine methylation by PRMT7 and stress-induced phosphorylation status of eIF2α at serine 51. Finally, we demonstrated that PRMT7 is required for eIF2α-dependent stress granule formation in the face of various cellular stresses. Altogether, our findings implicate PRMT7 as a novel mediator of eIF2α-dependent cellular stress response pathways.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

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