Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity

ABSTRACT In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2α (eIF2α). In addition, two of the three cellular eIF2α kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2α phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2α phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2α phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2α on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2α phosphorylation.

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The Potential Role of Protein Kinase R as a Regulator of Age-Related Neurodegeneration

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2021.638208 ◽

2021 ◽

Vol 13 ◽

Author(s):

Nicolás W. Martinez ◽

Felipe E. Gómez ◽

Soledad Matus

Keyword(s):

Protein Kinase ◽

Pathological Process ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Protein Kinase R ◽

Eukaryotic Translation ◽

Neurodegenerative Process ◽

Age Related ◽

Initiation Factor 2

There is a growing evidence describing a decline in adaptive homeostasis in aging-related diseases affecting the central nervous system (CNS), many of which are characterized by the appearance of non-native protein aggregates. One signaling pathway that allows cell adaptation is the integrated stress response (ISR), which senses stress stimuli through four kinases. ISR activation promotes translational arrest through the phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α) and the induction of a gene expression program to restore cellular homeostasis. However, depending on the stimulus, ISR can also induce cell death. One of the ISR sensors is the double-stranded RNA-dependent protein kinase [protein kinase R (PKR)], initially described as a viral infection sensor, and now a growing evidence supports a role for PKR on CNS physiology. PKR has been largely involved in the Alzheimer’s disease (AD) pathological process. Here, we reviewed the antecedents supporting the role of PKR on the efficiency of synaptic transmission and cognition. Then, we review PKR’s contribution to AD and discuss the possible participation of PKR as a player in the neurodegenerative process involved in aging-related pathologies affecting the CNS.

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

mBio ◽

10.1128/mbio.00976-20 ◽

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.

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Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

Journal of Virology ◽

10.1128/jvi.02714-15 ◽

2016 ◽

Vol 90 (8) ◽

pp. 3839-3848 ◽

Cited By ~ 26

Author(s):

Benjamin Ziehr ◽

Heather A. Vincent ◽

Nathaniel J. Moorman

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Viral Protein ◽

Hcmv Infection ◽

Initiation Factor ◽

Host Defenses ◽

Protein Kinase R ◽

Viral Protein Synthesis ◽

Eif2α Phosphorylation ◽

Hcmv Replication

ABSTRACTHuman cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication.IMPORTANCETo successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

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The Cellular Protein P58IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism

Journal of Virology ◽

10.1128/jvi.02151-06 ◽

2006 ◽

Vol 81 (5) ◽

pp. 2221-2230 ◽

Cited By ~ 54

Author(s):

Alan G. Goodman ◽

Jennifer A. Smith ◽

Siddharth Balachandran ◽

Olivia Perwitasari ◽

Sean C. Proll ◽

...

Keyword(s):

Influenza Virus ◽

Mammalian Cells ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Virus Protein ◽

Mrna Levels ◽

Viral Mrna ◽

Eif2α Phosphorylation ◽

In Cells

ABSTRACT We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58IPK, the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. P58IPK also inhibits PERK, an eIF2α kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58IPK to interact with and inhibit multiple eIF2α kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58IPK−/− mice, we demonstrated that the absence of P58IPK led to an increase in eIF2α phosphorylation and decreased influenza virus mRNA translation. The absence of P58IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58IPK target, PKR, the trends were reversed—eIF2α phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2α phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2α. Taken together, our results support a model in which P58IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

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EMBR-32. INTEGRATED STRESS RESPONSE PLAYS A PRO-SURVIVAL ROLE IN MYC-DRIVEN MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab090.049 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i12-i13

Author(s):

Sofya Langman ◽

Alberto Delaidelli ◽

Yue Zhou Huang ◽

Poul Sorensen

Keyword(s):

Stress Response ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Unfolded Proteins ◽

Treatment Regimens ◽

Eif2α Phosphorylation ◽

Cancer Cell Death

Abstract Medulloblastoma (MB) accounts for 20% of diagnosed brain tumors in children. Group 3 (G3) MB subtype is the most aggressive. Molecularly, G3 MB is characterized by MYC overexpression, which drives elevated mRNA translation in tumor cells. PERK is an eukaryotic translation initiation factor 2 (eIF2α) kinase that inhibits mRNA translation under endoplasmic reticulum (ER) stress conditions, such as in response to accumulation of unfolded proteins. When unfolded proteins accumulate in the ER, activated PERK phosphorylates eIF2α. This shuts down global translation and triggers integrated stress response (ISR) to help cells adapt through selective translation of mRNA encoding pro-survival proteins. High mRNA expression of PERK correlates with poor survival in G3 MB patients. In vitro, combination of ER or hypoxic stress with PERK knockdown induces apoptosis in MB cells. ISRIB is an ISR inhibitor that maintains translation rates despite eIF2α phosphorylation. Combining ISRIB with stress such as hypoxia induces apoptosis in MB cells and prevents accumulation of key ISR mediators such as ATF4. In addition, combination of ISRIB and hypoxia induces oxidative stress. Current G3 MB treatment regimens include vincristine, a known ISR inducer. Combination of ISRIB with vincristine amplifies vincristine-induced apoptosis, potentially suggesting novel therapeutic approach for MB. Our findings show that inhibition of ISR in G3 MB represents a powerful inducer of cancer cell death.

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GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis

Veterinary Research ◽

10.1186/s13567-021-01018-5 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Ting Zhu ◽

Xueli Jiang ◽

Hangkuo Xin ◽

Xiaohui Zheng ◽

Xiaonuan Xue ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Pseudorabies Virus ◽

De Novo ◽

Initiation Factor ◽

Protein Kinase R ◽

Eif2α Phosphorylation ◽

Phosphorylation Level ◽

Global Protein Synthesis ◽

De Novo Protein Synthesis

AbstractViruses have evolved multiple strategies to manipulate their host’s translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

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A critical review of translation initiation factor eIF2α kinases in plants - regulating protein synthesis during stress

Functional Plant Biology ◽

10.1071/fp12116 ◽

2012 ◽

Vol 39 (9) ◽

pp. 717 ◽

Cited By ~ 14

Author(s):

Tracey M. Immanuel ◽

David R. Greenwood ◽

Robin M. MacDiarmid

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Current Knowledge ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Α Subunit ◽

Eif2α Phosphorylation ◽

Phosphorylation Mechanism ◽

Eif2α Kinase

Eukaryotic cells must cope with environmental stress. One type of general stress response is the downregulation of protein synthesis in order to conserve cellular resources. Protein synthesis is mainly regulated at the level of mRNA translation initiation and when the α subunit of eukaryotic translation initiation factor 2 (eIF2) is phosphorylated, protein synthesis is downregulated. Although eIF2 has the same translation initiation function in all eukaryotes, it is not known whether plants downregulate protein synthesis via eIF2α phosphorylation. Similarly, although there is evidence that plants possess eIF2α kinases, it is not known whether they operate in a similar manner to the well characterised mammalian and yeast eIF2α kinases. Two types of eIF2α kinases have been reported in plants, yet the full understanding of the plant eIF2α phosphorylation mechanism is still lacking. Here we review the current knowledge of the eIF2α phosphorylation mechanism within plants and discuss plant eIF2α, plant eIF2α kinase GCN2 and the data supporting and contradicting the hypothesis that a functional orthologue for the eIF2α kinase PKR, is present and functional in plants.

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Translation Control by Protein Kinase R Restricts Minute Virus of Mice Infection: Role in Parvovirus Oncolysis

Journal of Virology ◽

10.1128/jvi.02188-09 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5043-5051 ◽

Cited By ~ 14

Author(s):

Iván Ventoso ◽

Juan J. Berlanga ◽

José M. Almendral

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Translational Control ◽

Translation Initiation Factor ◽

Protein Kinase R ◽

Minute Virus Of Mice ◽

Eif2α Phosphorylation ◽

3T3 Fibroblasts ◽

Minute Virus ◽

Nih 3T3

ABSTRACT The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKRo/o) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive PKRo/o cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2α phosphorylation and increased MVMp protein synthesis. I n vitro-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2α phosphorylation. In contrast, virus-permissive mouse (PKRo/o, BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2α phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.

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Murine Cytomegalovirus m142 and m143 Are both Required To Block Protein Kinase R-Mediated Shutdown of Protein Synthesis

Journal of Virology ◽

10.1128/jvi.00908-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10181-10190 ◽

Cited By ~ 55

Author(s):

Ralitsa S. Valchanova ◽

Marcus Picard-Maureau ◽

Matthias Budt ◽

Wolfram Brune

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Rna Binding ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Murine Cytomegalovirus ◽

Deletion Mutants ◽

Sequence Motifs ◽

Protein Kinase R ◽

Double Stranded Rna

ABSTRACT Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2α, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.

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