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

2006 ◽  
Vol 81 (5) ◽  
pp. 2221-2230 ◽  
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.

scholarly journals Efficient Translation of Rotavirus mRNA Requires Simultaneous Interaction of NSP3 with the Eukaryotic Translation Initiation Factor eIF4G and the mRNA 3′ End

2000 ◽  
Vol 74 (15) ◽  
pp. 7064-7071 ◽  
Author(s):  
Patrice Vende ◽  
Maria Piron ◽  
Nathalie Castagné ◽  
Didier Poncet
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrna ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3′-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3′ end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.

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 PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes

2003 ◽  
Vol 84 (12) ◽  
pp. 3263-3274 ◽  
Author(s):  
Idoia Burgui ◽  
Tomás Aragón ◽  
Juan Ortín ◽  
Amelia Nieto
Keyword(s):  
Influenza Virus ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Published Data ◽  
Ns1 Protein ◽  
Viral Mrnas ◽  
Independent Manner ◽  
Cellular Mrnas

It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 5′UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1–eIF4GI and PABP1–eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs.

scholarly journals Substrate recognition determinants of human eIF2α phosphatases

2021 ◽  
Author(s):  
George Hodgson ◽  
Antonina Andreeva ◽  
Anne Bertolotti
Keyword(s):  
Mammalian Cells ◽  
Biological Significance ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type ◽  
Binding Region ◽  
Antagonistic Action ◽  
Catalytic Subunits ◽  
Eif2α Phosphorylation ◽  
Cellular Defence

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.

scholarly journals Inhibition of a constitutive translation initiation factor 2α phosphatase, CReP, promotes survival of stressed cells

2003 ◽  
Vol 163 (4) ◽  
pp. 767-775 ◽  
Author(s):  
Céline Jousse ◽  
Seiichi Oyadomari ◽  
Isabel Novoa ◽  
Phoebe Lu ◽  
Yuhong Zhang ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Gene Encoding ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
Stressed Cells

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on serine 51 is effected by specific stress-activated protein kinases. eIF2α phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2α dephosphorylation, however, GADD34 mutant cells retain significant eIF2α-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2α. RNAi of this gene, which we named constitutive repressor of eIF2α phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2α-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2α dephosphorylation by targeting the CReP-protein–phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

Identification of GCD14 and GCD15, Novel Genes Required for Translational Repression of GCN4 mRNA in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1007-1020 ◽  
Author(s):  
Rafael Cuesta ◽  
Alan G Hinnebusch ◽  
Mercedes Tamame
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
New Genes ◽  
General Translation

Abstract In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid. The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions. The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B. To identify additional initiation factors in yeast, we characterized 65 spontaneously arising Gcd− mutants. In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15. Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control. The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation. We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency. The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13. Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

scholarly journals Cellular cap-binding proteins associate with influenza virus mRNAs

2011 ◽  
Vol 92 (7) ◽  
pp. 1627-1634 ◽  
Author(s):  
Katja Bier ◽  
Ashley York ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Nuclear Export ◽  
Binding Proteins ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
Ribonucleoprotein Complexes

The influenza virus RNA polymerase synthesizes three types of RNA: genomic vRNA, anti-genomic cRNA and mRNA. Both vRNA and cRNA are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mRNAs are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mRNA, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mRNA. We found that viral mRNA associates with the viral non-structural protein 1 (NS1), cellular poly(A)-binding protein 1 (PABP1), the 20 kDa subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eIF4E. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mRNP) complex. We propose a model in which viral mRNAs, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mRNAs for splicing, nuclear export and translation.

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs

1986 ◽  
Vol 6 (5) ◽  
pp. 1741-1750
Author(s):  
M G Katze ◽  
B M Detjen ◽  
B Safer ◽  
R M Krug
Keyword(s):  
Protein Synthesis ◽  
Influenza Virus ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
Double Stranded Rna ◽  
Selective Translation ◽  
Infected Cells

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

scholarly journals mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

2016 ◽  
Vol 473 (24) ◽  
pp. 4651-4664 ◽  
Author(s):  
Lyne Jossé ◽  
Jianling Xie ◽  
Christopher G. Proud ◽  
C. Mark Smales
Keyword(s):  
Protein Synthesis ◽  
Cell Growth ◽  
Recombinant Protein ◽  
Cell Lines ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cho Cell

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

scholarly journals Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus

2000 ◽  
Vol 20 (17) ◽  
pp. 6259-6268 ◽  
Author(s):  
Tomás Aragón ◽  
Susana de la Luna ◽  
Isabel Novoa ◽  
Luis Carrasco ◽  
Juan Ortín ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Binding ◽  
Binding Domain ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Virus Protein ◽  
Published Data ◽  
Ns1 Protein ◽  
Cellular Mrnas

ABSTRACT Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5′ untranslated region (5′ UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

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