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

2016 ◽  
Vol 90 (8) ◽  
pp. 3839-3848 ◽  
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.

scholarly journals The role of host eIF2α in viral infection

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuanzhi Liu ◽  
Mingshu Wang ◽  
Anchun Cheng ◽  
Qiao Yang ◽  
Ying Wu ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Viral Infection ◽  
Infection Process ◽  
Host Protein ◽  
Main Body ◽  
Protein Kinase R ◽  
Viral Protein Synthesis ◽  
Eif2α Phosphorylation

Abstract Background eIF2α is a regulatory node that controls protein synthesis initiation by its phosphorylation or dephosphorylation. General control nonderepressible-2 (GCN2), protein kinase R-like endoplasmic reticulum kinase (PERK), double-stranded RNA (dsRNA)-dependent protein kinase (PKR) and heme-regulated inhibitor (HRI) are four kinases that regulate eIF2α phosphorylation. Main body In the viral infection process, dsRNA or viral proteins produced by viral proliferation activate different eIF2α kinases, resulting in eIF2α phosphorylation, which hinders ternary tRNAMet-GTP-eIF2 complex formation and inhibits host or viral protein synthesis. The stalled messenger ribonucleoprotein (mRNP) complex aggregates under viral infection stress to form stress granules (SGs), which encapsulate viral RNA and transcription- and translation-related proteins, thereby limiting virus proliferation. However, many viruses have evolved a corresponding escape mechanism to synthesize their own proteins in the event of host protein synthesis shutdown and SG formation caused by eIF2α phosphorylation, and viruses can block the cell replication cycle through the PERK-eIF2α pathway, providing a favorable environment for their own replication. Subsequently, viruses can induce host cell autophagy or apoptosis through the eIF2α-ATF4-CHOP pathway. Conclusions This review summarizes the role of eIF2α in viral infection to provide a reference for studying the interactions between viruses and hosts.

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

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.

scholarly journals Essential Role for either TRS1 or IRS1 in Human Cytomegalovirus Replication

2009 ◽  
Vol 83 (9) ◽  
pp. 4112-4120 ◽  
Author(s):  
Emily E. Marshall ◽  
Craig J. Bierle ◽  
Wolfram Brune ◽  
Adam P. Geballe
Keyword(s):  
Protein Synthesis ◽  
Human Cytomegalovirus ◽  
Viral Infections ◽  
Productive Infection ◽  
Initiation Factor ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Viral Protein Synthesis ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACT Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2′-5′ oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[ΔI/ΔT]) has a severe replication defect. Infection with HCMV[ΔI/ΔT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[ΔI/ΔT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2α is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection.

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

2010 ◽  
Vol 84 (20) ◽  
pp. 10457-10466 ◽  
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.

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

2008 ◽  
Vol 83 (5) ◽  
pp. 2298-2309 ◽  
Author(s):  
Verena Krähling ◽  
David A. Stein ◽  
Martin Spiegel ◽  
Friedemann Weber ◽  
Elke Mühlberger
Keyword(s):  
Protein Kinase ◽  
Severe Acute Respiratory Syndrome ◽  
Chromatin Condensation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Beta Interferon ◽  
Reverse Transcription Pcr ◽  
Eif2α Phosphorylation

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.

scholarly journals Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 499 ◽  
Author(s):  
Shelby Powell Kesterson ◽  
Jeffery Ringiesn ◽  
Vikram N. Vakharia ◽  
Brian S. Shepherd ◽  
Douglas W. Leaman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Molecular Mechanisms ◽  
Freshwater Ecosystems ◽  
Innate Immune ◽  
Host Cells ◽  
Initiation Factor ◽  
Viral Hemorrhagic Septicemia Virus ◽  
Viral Protein Synthesis ◽  
Viral Hemorrhagic Septicemia

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

scholarly journals Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

2009 ◽  
Vol 84 (2) ◽  
pp. 1124-1138 ◽  
Author(s):  
Julianne L. Garrey ◽  
Yun-Young Lee ◽  
Hilda H. T. Au ◽  
Martin Bushell ◽  
Eric Jan
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Rna Virus ◽  
Virus Production ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
S2 Cells ◽  
Structural Protein ◽  
Viral Protein Synthesis ◽  
Paralysis Virus

ABSTRACT The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.

scholarly journals The Autophagy Protein ATG16L1 Is Required for Sindbis Virus-Induced eIF2α Phosphorylation and Stress Granule Formation

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 39
Author(s):  
Matthew Jefferson ◽  
Benjamin Bone ◽  
Jasmine L. Buck ◽  
Penny P. Powell
Keyword(s):  
Immune Response ◽  
Protein Synthesis ◽  
Innate Immune Response ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Binding Protein ◽  
Innate Immune ◽  
Stress Granule ◽  
Viral Protein Synthesis ◽  
Eif2α Phosphorylation

Sindbis virus (SINV) infection induces eIF2α phosphorylation, which leads to stress granule (SG) assembly. SINV infection also stimulates autophagy, which has an important role in controlling the innate immune response. The importance of autophagy to virus-induced translation arrest is not well understood. In this study, we show that the autophagy protein ATG16L1 not only regulates eIF2α phosphorylation and the translation of viral and antiviral proteins, but also controls SG assembly. Early in infection (2hpi), capsids were recruited by host factors Cytotoxic Granule-Associated RNA Binding Protein (TIA1), Y-box binding protein 1 (YBX1), and vasolin-containing protein 1 (VCP), to a single perinuclear body, which co-localized with the viral pattern recognition sensors, double stranded RNA-activated protein-kinase R (PKR) and RIG-I. By 6hpi, there was increased eIF2α phosphorylation and viral protein synthesis. However, in cells lacking the autophagy protein ATG16L1, SG assembly was inhibited and capsid remained in numerous small foci in the cytoplasm containing YBX1, TIA1 with RIG-I, and these persisted for over 8hpi. In the absence of ATG16L1, there was little phosphorylation of eIF2α and low levels of viral protein synthesis. Compared to wild type cells, there was potentiated interferon protein and interferon-stimulated gene (ISG) mRNA expression. These results show that ATG16L1 is required for maximum eIF2α phosphorylation, proper SG assembly into a single perinuclear focus, and for attenuating the innate immune response. Therefore, this study shows that, in the case of SINV, ATG16L1 is pro-viral, required for SG assembly and virus replication.

scholarly journals The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.

1994 ◽  
Vol 14 (4) ◽  
pp. 2331-2342 ◽  
Author(s):  
T G Lee ◽  
N Tang ◽  
S Thompson ◽  
J Miller ◽  
M G Katze
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Kinase ◽  
Immunoblot Analysis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Tetratricopeptide Repeat ◽  
Repeat Family ◽  
Viral Protein Synthesis ◽  
Initiation Factor 2

PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.

scholarly journals Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

2007 ◽  
Vol 82 (2) ◽  
pp. 828-839 ◽  
Author(s):  
Maria D. Gainey ◽  
Patrick J. Dillon ◽  
Kimberly M. Clark ◽  
Mary J. Manuse ◽  
Griffith D. Parks
Keyword(s):  
Protein Synthesis ◽  
Hela Cells ◽  
Viral Protein ◽  
Time Course ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
V Protein

ABSTRACT The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI− mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI− mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI− virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI− mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI− mutant was restored to ∼50% that of control HeLa cells. By contrast, the rates of P/V-CPI− viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI− mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI− mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

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