scholarly journals eIF2B-capturing viral protein NSs suppresses the integrated stress response

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kazuhiro Kashiwagi ◽  
Yuichi Shichino ◽  
Tatsuya Osaki ◽  
Ayako Sakamoto ◽  
Madoka Nishimoto ◽  
...  
Keyword(s):  
Stress Response ◽  
Hippocampal Neurons ◽  
Nonstructural Protein ◽  
Ribosome Profiling ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Ips Cell ◽  
Α Subunit ◽  
A Genome

AbstractVarious stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

scholarly journals eIF2B-capturing viral protein NSs suppresses the integrated stress response

2021 ◽  
Author(s):  
Kazuhiro Kashiwagi ◽  
Yuichi Shichino ◽  
Tatsuya Osaki ◽  
Ayako Sakamoto ◽  
Madoka Nishimoto ◽  
...  
Keyword(s):  
Stress Response ◽  
Cultured Cells ◽  
Nonstructural Protein ◽  
Ribosome Profiling ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Neuroprotective Effects ◽  
Α Subunit ◽  
A Genome

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotides on unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. Our cryo-electron microscopy (cryo-EM) analysis revealed that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B exhibits normal nucleotide exchange activity even in the presence of eIF2(αP). A genome-wide ribosome profiling analysis clarified that SFSV NSs in human cultured cells attenuates the ISR. Furthermore, SFSV NSs exhibited neuroprotective effects against the ISR-inducing stress. Since the ISR inhibition is beneficial in various neurological disease models, SFSV NSs is promising as a therapeutic ISR inhibitor.

scholarly journals eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

2020 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

scholarly journals eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael Schoof ◽  
Morgane Boone ◽  
Lan Wang ◽  
Rosalie Lawrence ◽  
Adam Frost ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Sites ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

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

2015 ◽  
Vol 113 (2) ◽  
pp. E117-E126 ◽  
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.

scholarly journals Small molecule ISRIB suppresses the integrated stress response within a defined window of activation

2019 ◽  
Vol 116 (6) ◽  
pp. 2097-2102 ◽  
Author(s):  
Huib H. Rabouw ◽  
Martijn A. Langereis ◽  
Aditya A. Anand ◽  
Linda J. Visser ◽  
Raoul J. de Groot ◽  
...  
Keyword(s):  
Stress Response ◽  
Side Effects ◽  
Small Molecule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Poly I:C ◽  
Critical Threshold ◽  
Therapeutic Effects ◽  
Integrated Stress Response ◽  
Α Subunit

Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2•GDP to active eIF2•GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.

scholarly journals eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jennifer Deborah Wuerth ◽  
Matthias Habjan ◽  
Markus Kainulainen ◽  
Besim Berisha ◽  
Damien Bertheloot ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Nonstructural Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mammalian Host ◽  
Eukaryotic Initiation Factor ◽  
Α Subunit ◽  
Immune Factor

ABSTRACT RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α. IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

scholarly journals Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J. Zachery Cogan ◽  
Rosalie E. Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

scholarly journals Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection

2021 ◽  
Vol 22 (11) ◽  
pp. 5646
Author(s):  
Qing Chun Zhu ◽  
Shumin Li ◽  
Li Xia Yuan ◽  
Rui Ai Chen ◽  
Ding Xiang Liu ◽  
...  
Keyword(s):  
Stress Response ◽  
Mrna Expression ◽  
Translation Initiation Factor ◽  
Interleukin 8 ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Activator Protein 1 ◽  
Diarrhea Virus ◽  
Cytokines And Chemokines ◽  
Coronavirus Infection

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

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