Reovirus Induces and Benefits from an Integrated Cellular Stress Response

ABSTRACT Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58IPK, the cellular inhibitor of the eukaryotic initiation factor 2α (eIF2α) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2α phosphorylation and unphosphorylated eIF2α is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2α phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2α kinases and phosphorylatable eIF2α. When eIF2α is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2α phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2α phosphorylation facilitates reovirus replication in two ways—first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

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Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions

Journal of Virology ◽

10.1128/jvi.00459-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7268-7284 ◽

Cited By ~ 35

Author(s):

Emily V. Nelson ◽

Kristina M. Schmidt ◽

Laure R. Deflubé ◽

Sultan Doğanay ◽

Logan Banadyga ◽

...

Keyword(s):

Stress Response ◽

Ebola Virus ◽

Cellular Stress ◽

Stress Granule ◽

Cellular Stress Response ◽

Initiation Factor ◽

Ebov Infection ◽

Infected Cells ◽

Translational Arrest ◽

Viral Inclusions

ABSTRACTA hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions.IMPORTANCEAlthough progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

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Junín virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2α phosphorylation

Journal of General Virology ◽

10.1099/vir.0.033407-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2889-2899 ◽

Cited By ~ 30

Author(s):

Florencia N. Linero ◽

María G. Thomas ◽

Graciela L. Boccaccio ◽

Luis A. Scolaro

Keyword(s):

Stress Response ◽

Matrix Protein ◽

Acute Infection ◽

Protein Translation ◽

Vero Cells ◽

Cellular Stress Response ◽

Protein Z ◽

Granule Formation ◽

Eif2α Phosphorylation ◽

Infected Cells

Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.

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An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1514076113 ◽

2015 ◽

Vol 113 (2) ◽

pp. E117-E126 ◽

Cited By ~ 23

Author(s):

Aude De Gassart ◽

Bojan Bujisic ◽

Léa Zaffalon ◽

Laurent A. Decosterd ◽

Antonia Di Micco ◽

...

Keyword(s):

Stress Response ◽

Stress Responses ◽

Translation Initiation Factor ◽

Hiv Protease ◽

Initiation Factor ◽

Hiv Protease Inhibitors ◽

Integrated Stress Response ◽

Α Subunit ◽

Eif2α Phosphorylation ◽

Program Characteristic

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.

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Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health

Biological Chemistry ◽

10.1515/hsz-2019-0385 ◽

2020 ◽

Vol 401 (9) ◽

pp. 1005-1018

Author(s):

Laura M. Jones ◽

Yannic Chen ◽

Patricija van Oosten-Hawle

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Stress Response ◽

Stress Responses ◽

Stress Condition ◽

Physiological Responses ◽

Defense Responses ◽

Cellular Stress Response ◽

Proteotoxic Stress ◽

Eukaryotic Organisms

AbstractEukaryotic organisms have evolved complex and robust cellular stress response pathways to ensure maintenance of proteostasis and survival during fluctuating environmental conditions. Highly conserved stress response pathways can be triggered and coordinated at the cell-autonomous and cell-nonautonomous level by proteostasis transcription factors, including HSF1, SKN-1/NRF2, HIF1, and DAF-16/FOXO that combat proteotoxic stress caused by environmental challenges. While these transcription factors are often associated with a specific stress condition, they also direct “noncanonical” transcriptional programs that serve to integrate a multitude of physiological responses required for development, metabolism, and defense responses to pathogen infections. In this review, we outline the established function of these key proteostasis transcription factors at the cell-autonomous and cell-nonautonomous level and discuss a newly emerging stress responsive transcription factor, PQM-1, within the proteostasis network. We look beyond the canonical stress response roles of proteostasis transcription factors and highlight their function in integrating different physiological stimuli to maintain cytosolic organismal proteostasis.

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Relevance of oxidative stress in inhibition of eIF2 alpha phosphorylation and stress granules formation during Usutu virus infection

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009072 ◽

2021 ◽

Vol 15 (1) ◽

pp. e0009072

Author(s):

Ana-Belén Blázquez ◽

Miguel A. Martín-Acebes ◽

Teresa Poderoso ◽

Juan-Carlos Saiz

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Stress Response ◽

Stress Responses ◽

Stress Granules ◽

Initiation Factor ◽

Potential Health ◽

Usutu Virus ◽

Cellular Environment ◽

Infected Cells

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile, Japanese encephalitis, Zika, and dengue viruses. USUV emerged in 1996 in Europe, where quickly spread across the continent causing a considerable number of bird deaths and varied neurological disorders in humans, including encephalitis, meningoencephalitis, or facial paralysis, thus warning about USUV as a potential health threat. USUV replication takes place on the endoplasmic reticulum (ER) of infected cells, inducing ER stress and resulting in the activation of stress-related cellular pathways collectively known as the integrated stress response (ISR). The alpha subunit of the eukaryotic initiation factor eIF2 (eIF2α), the core factor in this pathway, is phosphorylated by stress activated kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated inhibitor kinase (HRI), and general control non-repressed 2 kinase (GCN2). Its phosphorylation results, among others, in the downstream inhibition of translation with accumulation of discrete foci in the cytoplasm termed stress granules (SGs). Our results indicated that USUV infection evades cellular stress response impairing eIF2α phosphorylation and SGs assembly induced by treatment with the HRI activator ArsNa. This protective effect was related with oxidative stress responses in USUV-infected cells. Overall, these results provide new insights into the complex connections between the stress response and flavivirus infection in order to maintain an adequate cellular environment for viral replication.

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Hypoxic Inhibition of Nonsense-Mediated RNA Decay Regulates Gene Expression and the Integrated Stress Response

Molecular and Cellular Biology ◽

10.1128/mcb.02284-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3729-3741 ◽

Cited By ~ 131

Author(s):

Lawrence B. Gardner

Keyword(s):

Gene Expression ◽

Stress Response ◽

Cellular Response ◽

Cellular Stress Response ◽

Initiation Factor ◽

Rna Decay ◽

Α Subunit ◽

Eif2α Phosphorylation ◽

Cellular Mrnas ◽

Nonsense Mediated Rna Decay

ABSTRACT Nonsense-mediated RNA decay (NMD) rapidly degrades both mutated mRNAs and nonmutated cellular mRNAs in what is thought to be a constitutive fashion. Here we demonstrate that NMD is inhibited in hypoxic cells and that this inhibition is dependent on phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). eIF2α phosphorylation is known to promote translational and transcriptional up-regulation of genes important for the cellular response to stress. We show that the mRNAs of several of these stress-induced genes are NMD targets and that the repression of NMD stabilizes these mRNAs, thus demonstrating that the inhibition of NMD augments the cellular stress response. Furthermore, hypoxia-induced formation of cytoplasmic stress granules is also dependent on eIF2α phosphorylation, and components of the NMD pathway are relocalized to these granules in hypoxic cells, providing a potential mechanism for the hypoxic inhibition of NMD. Our demonstration that NMD is inhibited in hypoxic cells reveals that the regulation of NMD can dynamically alter gene expression and also establishes a novel mechanism for hypoxic gene regulation.

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Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

Cell Death and Disease ◽

10.1038/s41419-020-03324-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ai-Ling Tian ◽

Qi Wu ◽

Peng Liu ◽

Liwei Zhao ◽

Isabelle Martins ◽

...

Keyword(s):

Stress Response ◽

Immune Responses ◽

Initiation Factor ◽

Integrated Stress Response ◽

Serine Residue ◽

Eif2α Phosphorylation ◽

Lysosomotropic Agents ◽

Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

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Flavonoids: Potential Candidates for the Treatment of Neurodegenerative Disorders

Biomedicines ◽

10.3390/biomedicines9020099 ◽

2021 ◽

Vol 9 (2) ◽

pp. 99

Author(s):

Shweta Devi ◽

Vijay Kumar ◽

Sandeep Kumar Singh ◽

Ashish Kant Dubey ◽

Jong-Joo Kim

Keyword(s):

Stress Response ◽

Stress Responses ◽

Neurodegenerative Disorders ◽

Cell Damage ◽

Cellular Stress ◽

Clinical Manifestations ◽

Cellular Stress Response ◽

Dramatic Rise ◽

Cellular Stress Responses

Neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), are the most concerning disorders due to the lack of effective therapy and dramatic rise in affected cases. Although these disorders have diverse clinical manifestations, they all share a common cellular stress response. These cellular stress responses including neuroinflammation, oxidative stress, proteotoxicity, and endoplasmic reticulum (ER)-stress, which combats with stress conditions. Environmental stress/toxicity weakened the cellular stress response which results in cell damage. Small molecules, such as flavonoids, could reduce cellular stress and have gained much attention in recent years. Evidence has shown the potential use of flavonoids in several ways, such as antioxidants, anti-inflammatory, and anti-apoptotic, yet their mechanism is still elusive. This review provides an insight into the potential role of flavonoids against cellular stress response that prevent the pathogenesis of neurodegenerative disorders.

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

Journal of Virology ◽

10.1128/jvi.01010-19 ◽

2019 ◽

Vol 93 (19) ◽

Cited By ~ 4

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.

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Protein Kinase R Is Responsible for the Phosphorylation of eIF2α in Rotavirus Infection

Journal of Virology ◽

10.1128/jvi.00625-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10457-10466 ◽

Cited By ~ 50

Author(s):

Margarito Rojas ◽

Carlos F. Arias ◽

Susana López

Keyword(s):

Protein Synthesis ◽

Kinase Domain ◽

Cell Protein ◽

Protein Kinase R ◽

Viral Protein Synthesis ◽

Α Subunit ◽

Rotavirus Infection ◽

Eif2α Phosphorylation ◽

Infected Cells ◽

Interferon System

ABSTRACT The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the α subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2α becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2α in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2α was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2α phosphorylation.

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