Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.     

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer''s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.     

Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production

Cells can undergo two alternative fates following exposure to environmental stress: they either induce apoptosis or inhibit apoptosis and then repair the stress-induced alterations. These processes minimize cell loss and prevent the survival of cells with aberrant DNA and protein alterations. These two alternative fates are partly controlled by stress granules (SGs). While arsenite, hypoxia, and heat shock induce the formation of SGs that inhibit apoptosis, X-ray irradiation and genotoxic drugs do not induce SGs, and they are more prone to trigger apoptosis. However, it is unclear precisely how SGs control apoptosis. This study found that SGs suppress the elevation of reactive oxygen species (ROS), and this suppression is essential for inhibiting ROS-dependent apoptosis. This antioxidant activity of SGs is controlled by two SG components, GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and ubiquitin-specific protease 10 (USP10). G3BP1 elevates the steady-state ROS level by inhibiting the antioxidant activity of USP10. However, following exposure to arsenite, G3BP1 and USP10 induce the formation of SGs, which uncovers the antioxidant activity of USP10. We also found that the antioxidant activity of USP10 requires the protein kinase activity of ataxia telangiectasia mutated (ATM). This work reveals that SGs are critical redox regulators that control cell fate under stress conditions.     

Regulation of AR mRNA translation in response to acute AR pathway inhibition

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid–liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.     

Stress granules are dispensable for mRNA stabilization during cellular stress

During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.     

YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.     

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.     

Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase

Mammalian cells form dynamic cytoplasmic mRNA stress granules (SGs) in response to environmental stresses including viral infections. SGs are involved in regulating host mRNA function and metabolism, although their precise role during viral infection is unknown. SGs are thought to assemble based on functions of the RNA-binding proteins TIA-1/TIAR or Ras-GAP SH3 domain-binding protein (G3BP). Here, we investigated the relationship between a prototypical plus-strand RNA virus and SGs. Early during poliovirus infection, SG formation is induced, but as infection proceeds this ability is lost, and SGs disperse. Infection resulted in cleavage of G3BP, but not TIA-1 or TIAR, by poliovirus 3C proteinase. Expression of a cleavage-resistant G3BP restored SG formation during poliovirus infection and significantly inhibited virus replication. These results elucidate a mechanism for viral interference with mRNP metabolism and gene regulation and support a critical role of G3BP in SG formation and restriction of virus replication.     

In silico identification of MicroRNAs targeting the key nucleator of stress granules,G3BP: Promising therapeutics for SARS-CoV-2 infection

Stress granules (SGs) are non-membrane ribonucleoprotein condensates formed in response to environmental stress conditions via liquid–liquid phase separation (LLPS). SGs are involved in the pathogenesis of aging and aging-associated diseases, cancers, viral infection, and several other diseases. GTPase-activating protein (SH3 domain)-binding protein 1 and 2 (G3BP1/2) is a key component and commonly used marker of SGs. Recent studies have shown that SARS-CoV-2 nucleocapsid protein via sequestration of G3BPs inhibits SGs formation in the host cells. In this study, we have identified putative miRNAs targeting G3BP in search of modulators of the G3BP expression. These miRNAs could be considered as new therapeutic targets against COVID-19 infection via the regulation of SG assembly and dynamics.     

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Tudor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions

SGs are mRNA containing cytoplasmic structures that are assembled in response to stress. Tudor-SN protein is a ubiquitously expressed protein. Here, Tudor-SN protein was found to physiologically interact with G3BP, which is the marker and effector of SG. The kinetics of the assembly of SGs in the living cells demonstrated that Tudor-SN co-localizes with G3BP and is recruited to the same SGs in response to different stress stimuli. Knockdown of endogenous Tudor-SN did not inhibit the formation of SGs, but retarded the aggregation of small SGs into large SGs. Thus Tudor-SN may not be an initiator as essential as G3BP for the formation of SGs, but affects the aggregation of SGs. These findings identify Tudor-SN as a novel component of SGs.

Structured summary

MINT-7968768, MINT-7968779: Tudor-SN (uniprotkb:Q7KZF4) physically interacts (MI:0915) with G3BP (uniprotkb:Q13283) by anti bait coimmunoprecipitation (MI:0006) MINT-7968800: Tudor-SN (uniprotkb:Q7KZF4) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7968789: Tudor-SN (uniprotkb:Q7KZF4) and G3BP (uniprotkb:Q13283) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Human Tudor staphylococcal nuclease (Tudor-SN) protein modulates the kinetics of AGTR1-3′UTR granule formation

Human Tudor staphylococcal nuclease (Tudor-SN) interacts with the G3BP protein and is recruited into stress granules (SGs), the main type of discrete RNA-containing cytoplasmic foci structure that is formed under stress conditions. Here, we further demonstrate that Tudor-SN binds and co-localizes with AGTR1-3′UTR (3′-untranslated region of angiotensin II receptor, type 1 mRNA) into SG. Tudor-SN plays an important role in the assembly of AGTR1-3′UTR granules. Moreover, endogenous Tudor-SN knockdown can decrease the recovery kinetics of AGTR1-3′UTR granules. Collectively, our data indicate that Tudor-SN modulates the kinetics of AGTR1-3′UTR granule formation, which provides an additional biological role of Tudor-SN in RNA metabolism during stress.     

Stress granules,RNA-binding proteins and polyglutamine diseases: too much aggregation?

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context.Subject terms: Neuroscience, Neurological disorders     

应激颗粒：细胞调控病毒感染的重要策略

真核细胞受到热休克、氧化应激、营养缺乏或者病毒感染等外界压力的刺激下会诱导一系列的应答反应,如形成应激颗粒(stress granule,SG),从而使细胞能更好地适应环境压力。SG作为胞浆中翻译起始复合物的聚集产物,在细胞的基因表达和稳态中发挥着重要的作用。病毒感染是诱导SG形成的条件之一,病毒侵入宿主细胞后会"借用"宿主的翻译机制完成自己的生命周期,宿主为了抵抗病毒的侵略而暂停翻译形成SG。本文对SG的产生及功能,SG与病毒的相互作用以及SG与病毒诱导的先天性免疫的关系等方面进行了综述,以期为进一步研究抗病毒策略提供方向。