THE TRANSCRIPTIONAL RESPONSE TO OXIDATIVE STRESS IS INDEPENDENT OF STRESS-GRANULE FORMATION

ABSTRACTCells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein, and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this issue, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Sodium-arsenite stress for 3 hours predominantly resulted in the induction or upregulation of stress-responsive mRNAs whose levels peaked during cell recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for protein chaperones, including HSP70 and low molecular-weight heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced prominent cytoplasmic stress granules that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/ Rasputin protein inhibited stress-granule assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced effects on gene expression appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

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The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation

Molecular Biology of the Cell ◽

10.1091/mbc.e21-08-0418 ◽

2022 ◽

Author(s):

Amanjot Singh ◽

Arvind Reddy Kandi ◽

Deepa Jayaprakashappa ◽

Guillaume Thuery ◽

Devam J Purohit ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Transcriptional Response ◽

Granule Formation ◽

Unfolded Protein ◽

Transcriptional Changes ◽

Translational Arrest ◽

Shock Proteins ◽

Gene Expression Alterations

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

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Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules

Journal of Virology ◽

10.1128/jvi.01168-19 ◽

2019 ◽

Vol 94 (3) ◽

Cited By ~ 4

Author(s):

Wanda Christ ◽

Janne Tynell ◽

Jonas Klingström

Keyword(s):

Protein Kinase ◽

Host Cell ◽

Stress Responses ◽

Stress Granules ◽

Cell Stress ◽

Stress Granule ◽

Hantavirus Infection ◽

Stress Signaling ◽

Protein Kinase R ◽

Granule Formation

ABSTRACT Virus infection frequently triggers host cell stress signaling resulting in translational arrest; as a consequence, many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study, we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs) but does so in only a limited proportion of infected cells. Our data indicate some cell type-specific and hantavirus species-specific variability in SG prevalence and show SG formation to be dependent on the activation of protein kinase R (PKR). Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like endoplasmic reticulum (ER) kinase (PERK). IMPORTANCE Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

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

Journal of Virology ◽

10.1128/jvi.01320-09 ◽

2009 ◽

Vol 84 (7) ◽

pp. 3654-3665 ◽

Cited By ~ 82

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.

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Infectious bronchitis virus regulates cellular stress granule signaling

10.1101/819482 ◽

2019 ◽

Author(s):

Matthew J. Brownsword ◽

Nicole Doyle ◽

Michèle Brocard ◽

Nicolas Locker ◽

Helena J. Maier

Keyword(s):

Stress Responses ◽

Infectious Bronchitis Virus ◽

Cellular Stress ◽

Stress Granules ◽

Stress Granule ◽

Granule Formation ◽

Translation Machinery ◽

Infectious Bronchitis ◽

Eif2α Phosphorylation ◽

Cellular Translation

AbstractViruses must hijack cellular translation machinery to efficiently express viral genes. In many cases, this is impeded by cellular stress responses. These stress responses swiftly relocate and repurpose translation machinery, resulting in global inhibition of translation and the aggregation of stalled 48S mRNPs into cytoplasmic foci called stress granules. This results in translational silencing of all mRNAs excluding those beneficial for the cell to resolve the specific stress. For example, expression of antiviral factors is maintained during viral infection. Here we investigated stress granule regulation by Gammacoronavirus infectious bronchitis virus (IBV), which causes the economically important poultry disease, infectious bronchitis. Interestingly, we found that IBV is able to inhibit multiple cellular stress granule signaling pathways whilst at the same time IBV replication also results in induction of seemingly canonical stress granules in a proportion of infected cells. Moreover, IBV infection uncouples translational repression and stress granule formation and both processes are independent of eIF2α phosphorylation. These results provide novel insights into how IBV modulates cellular translation and antiviral stress signaling.

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Novel Antioxidant Therapy with the Immediate Precursor to Glutathione, γ-Glutamylcysteine (GGC), Ameliorates LPS-Induced Cellular Stress in In Vitro 3D-Differentiated Airway Model from Primary Cystic Fibrosis Human Bronchial Cells

Antioxidants ◽

10.3390/antiox9121204 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1204

Author(s):

Chris K. Hewson ◽

Alexander Capraro ◽

Sharon L. Wong ◽

Elvis Pandzic ◽

Ling Zhong ◽

...

Keyword(s):

Oxidative Stress ◽

Cystic Fibrosis ◽

Stress Responses ◽

Therapeutic Potential ◽

Membrane Integrity ◽

The Body ◽

Stress Granule ◽

Cellular Respiration ◽

Granule Formation ◽

Lps Challenge

Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.

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Novel antioxidant therapy with the immediate precursor to glutathione, γ-glutamylcysteine (GGC), ameliorates LPS-induced cellular stress in an in vitro cystic fibrosis model

10.1101/2020.05.27.119990 ◽

2020 ◽

Author(s):

Chris K Hewson ◽

Alexander Capraro ◽

Sharon L Wong ◽

Elvis Pandzic ◽

Bentotage S.M Fernando ◽

...

Keyword(s):

Oxidative Stress ◽

Cystic Fibrosis ◽

Cell Viability ◽

Stress Responses ◽

Therapeutic Potential ◽

Membrane Integrity ◽

Stress Granule ◽

Cellular Respiration ◽

Granule Formation ◽

Lps Challenge

AbstractIntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, γ-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed.MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed.ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful.ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

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Infectious Bronchitis Virus Regulates Cellular Stress Granule Signaling

Viruses ◽

10.3390/v12050536 ◽

2020 ◽

Vol 12 (5) ◽

pp. 536

Author(s):

Matthew J. Brownsword ◽

Nicole Doyle ◽

Michèle Brocard ◽

Nicolas Locker ◽

Helena J. Maier

Keyword(s):

Stress Responses ◽

Infectious Bronchitis Virus ◽

Cellular Stress ◽

Stress Granules ◽

Stress Granule ◽

Granule Formation ◽

Infectious Bronchitis ◽

Eif2α Phosphorylation ◽

Cellular Translation ◽

Cellular Stress Responses

Viruses must hijack cellular translation machinery to express viral genes. In many cases, this is impeded by cellular stress responses. These stress responses result in the global inhibition of translation and the storage of stalled mRNAs, into RNA-protein aggregates called stress granules. This results in the translational silencing of the majority of mRNAs excluding those beneficial for the cell to resolve the specific stress. For example, the expression of antiviral factors is maintained during viral infection. Here we investigated stress granule regulation by Gammacoronavirus infectious bronchitis virus (IBV), which causes the economically important poultry disease, infectious bronchitis. Interestingly, we found that IBV is able to inhibit multiple cellular stress granule signaling pathways, whilst at the same time, IBV replication also results in the induction of seemingly canonical stress granules in a proportion of infected cells. Moreover, IBV infection uncouples translational repression and stress granule formation and both processes are independent of eIF2α phosphorylation. These results provide novel insights into how IBV modulates cellular translation and antiviral stress signaling.

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Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus

Journal of Virology ◽

10.1128/jvi.00403-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Guillaume Beauclair ◽

Felix Streicher ◽

Maxime Chazal ◽

Daniela Bruni ◽

Sarah Lesage ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Rna Virus ◽

Stress Granules ◽

Cytokine Response ◽

Fever Virus ◽

Stress Granule ◽

Proinflammatory Response ◽

Granule Formation ◽

Proinflammatory Mediators

ABSTRACT Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response. IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

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The Transcriptional Response to a Peroxisome Proliferator-activated Receptor α Agonist Includes Increased Expression of Proteome Maintenance Genes

Journal of Biological Chemistry ◽

10.1074/jbc.m409347200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52390-52398 ◽

Cited By ~ 64

Author(s):

Steven P. Anderson ◽

Paul Howroyd ◽

Jie Liu ◽

Xun Qian ◽

Rainer Bahnemann ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Transcriptional Response ◽

Lipid Homeostasis ◽

Peroxisome Proliferator ◽

Wild Type ◽

Peroxisome Proliferator Activated Receptor ◽

Proteasomal Genes ◽

Oxidative Stress Responses

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα), in addition to regulating lipid homeostasis, controls the level of tissue damage after chemical or physical stress. To determine the role of PPARα in oxidative stress responses, we examined damage after exposure to chemicals that increase oxidative stress in wild-type or PPARα-null mice. Primary hepatocytes from wild-type but not PPARα-null mice pretreated with the PPAR pan-agonist WY-14,643 (WY) were protected from damage to cadmium and paraquat. The livers from intact wild-type but not PPARα-null mice were more resistant to damage after carbon tetrachloride treatment. To determine the molecular basis of the protection by PPARα, we identified by transcript profiling genes whose expression was altered by a 7-day exposure to WY in wild-type and PPARα-null mice. Of the 815 genes regulated by WY in wild-type mice (p≤ 0.001; ≥1.5-fold or ≤-1.5-fold), only two genes were regulated similarly by WY in PPARα-null mice. WY increased expression of stress modifier genes that maintain the health of the proteome, including those that prevent protein aggregation (heat stress-inducible chaperones) and eliminate damaged proteins (proteasome components). Although the induction of proteasomal genes significantly overlapped with those regulated by 1,2-dithiole-3-thione, an activator of oxidant-inducible Nrf2, WY increased expression of proteasomal genes independently of Nrf2. Thus, PPARα controls the vast majority of gene expression changes after exposure to WY in the mouse liver and protects the liver from oxidant-induced damage, possibly through regulation of a distinct set of proteome maintenance genes.

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Multiple Pathways Differentially Regulate Global Oxidative Stress Responses in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e07-08-0735 ◽

2008 ◽

Vol 19 (1) ◽

pp. 308-317 ◽

Cited By ~ 125

Author(s):

Dongrong Chen ◽

Caroline R.M. Wilkinson ◽

Stephen Watt ◽

Christopher J. Penkett ◽

W. Mark Toone ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Hydrogen Peroxide ◽

Oxidative Damage ◽

Stress Responses ◽

Zinc Finger Protein ◽

Transcriptional Response ◽

Minor Role ◽

Transcriptional Responses ◽

Regulatory Pathways

Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1–like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.

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