scholarly journals Cleavage of Eukaryotic Translation Initiation Factor 4GII within Foot-and-Mouth Disease Virus-Infected Cells: Identification of the L-Protease Cleavage Site In Vitro

2004 ◽  
Vol 78 (7) ◽  
pp. 3271-3278 ◽  
Author(s):  
Alessandra Gradi ◽  
Nicole Foeger ◽  
Rebecca Strong ◽  
Yuri V. Svitkin ◽  
Nahum Sonenberg ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Foot And Mouth Disease ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Mouth Disease Virus ◽  
Eukaryotic Translation Initiation ◽  
Infected Cells ◽  
Mutant Forms

ABSTRACT Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (Lpro) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2Apro). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV Lpro specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2Apro in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV Lpro in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV Lpro and rhinovirus 2Apro enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.

scholarly journals Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression

2016 ◽  
Vol 90 (24) ◽  
pp. 11106-11121 ◽  
Author(s):  
Zixiang Zhu ◽  
Guoqing Wang ◽  
Fan Yang ◽  
Weijun Cao ◽  
Ruoqing Mao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Foot And Mouth Disease ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Mouth Disease Virus ◽  
2B Protein ◽  
Eukaryotic Translation Initiation ◽  
Cellular Apoptosis

ABSTRACTThe role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lproand 3Cproare viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein.IMPORTANCEThis study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

scholarly journals Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

1997 ◽  
Vol 17 (12) ◽  
pp. 6876-6886 ◽  
Author(s):  
S Z Tarun ◽  
A B Sachs
Keyword(s):  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

scholarly journals Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

scholarly journals Implications of the Use of Eukaryotic Translation Initiation Factor 5A (eIF5A) for Prognosis and Treatment of Hepatocellular Carcinoma

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Felix H. Shek ◽  
Sarwat Fatima ◽  
Nikki P. Lee
Keyword(s):  
Hepatocellular Carcinoma ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Of Translation ◽  
Eukaryotic Translation Initiation ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is a primary liver malignancy and accounts for most of the total liver cancer cases. Lack of treatment options and late diagnosis contribute to high mortality rate of HCC. In eukaryotes, translation of messenger RNA (mRNA) to protein is a key process in protein biosynthesis in which initiation of translation involves interaction of different eukaryotic translation initiation factors (eIFs), ribosome subunits and mRNAs. Eukaryotic translation initiation factor 5A (eIF5A) is one of the eIFs involved in translation initiation and eIF5A2, one of its isoforms, is upregulated in various cancers including HCC as a result of chromosomal instability, where it resides. In HCC, eIF5A2 expression is associated with adverse prognosis such as presence of tumor metastasis and venous infiltration. Based on eIF5A2 functional studies, suppressing eIF5A2 expression by short interfering RNA alleviates the tumorigenic properties of HCC cellsin vitrowhile ectopic expression of eIF5A2 enhances the aggressiveness of HCC cellsin vivoandin vitroby inducing epithelial-mesenchymal transition. In conclusion, eIF5A2 is a potential prognostic marker as well as a therapeutic target for HCC.

scholarly journals Generation of Multiple Isoforms of Eukaryotic Translation Initiation Factor 4GI by Use of Alternate Translation Initiation Codons

2002 ◽  
Vol 22 (13) ◽  
pp. 4499-4511 ◽  
Author(s):  
Marshall P. Byrd ◽  
Miguel Zamora ◽  
Richard E. Lloyd
Keyword(s):  
Translation Initiation ◽  
Cdna Clone ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Ires Element

ABSTRACT Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5′ end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5′ sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5′ untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

scholarly journals Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

2016 ◽  
Author(s):  
Colin Echeverría Aitken ◽  
Petra Beznosková ◽  
Vladislava Vlčkova ◽  
Wen-Ling Chiu ◽  
Fujun Zhou ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Functional Variants ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3

AbstractEukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2·GTP·Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncover a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

scholarly journals Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Colin Echeverría Aitken ◽  
Petra Beznosková ◽  
Vladislava Vlčkova ◽  
Wen-Ling Chiu ◽  
Fujun Zhou ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Functional Variants ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

scholarly journals mRNA Decapping in Yeast Requires Dissociation of the Cap Binding Protein, Eukaryotic Translation Initiation Factor 4E

2000 ◽  
Vol 20 (21) ◽  
pp. 7933-7942 ◽  
Author(s):  
David C. Schwartz ◽  
Roy Parker
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Sensitive Allele

ABSTRACT A major pathway of eukaryotic mRNA turnover occurs by deadenylation-dependent decapping that exposes the transcript to 5′→3′ exonucleolytic degradation. A critical step in this pathway is decapping, since removal of the cap structure permits 5′→3′ exonucleolytic digestion. Based on alterations in mRNA decay rate from strains deficient in translation initiation, it has been proposed that the decapping rate is modulated by a competition between the cytoplasmic cap binding complex, which promotes translation initiation, and the decapping enzyme, Dcp1p. In order to test this model directly, we examined the functional interaction of Dcp1p and the cap binding protein, eukaryotic translation initiation factor 4E (eIF4E), in vitro. These experiments indicated that eIF4E is an inhibitor of Dcp1p in vitro due to its ability to bind the 5′ cap structure. In addition, we demonstrate that in vivo a temperature-sensitive allele of eIF4E (cdc33-42) suppressed the decapping defect of a partial loss-of-function allele of DCP1. These results argue that dissociation of eIF4E from the cap structure is required before decapping. Interestingly, the temperature-sensitive allele of eIF4E does not suppress the decapping defect seen in strains lacking the decapping activators, Lsm1p and Pat1p. This indicates that these activators of decapping affect a step in mRNA turnover distinct from the competition between Dcp1 and eIF4E.

scholarly journals SUI1/p16 is required for the activity of eukaryotic translation initiation factor 3 in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (5) ◽  
pp. 2307-2313 ◽  
Author(s):  
T Naranda ◽  
S E MacMillan ◽  
T F Donahue ◽  
J W Hershey
Keyword(s):  
Translation Initiation ◽  
In Vitro Assay ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Vitro Assay ◽  
Eukaryotic Translation ◽  
Initiator Codon ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3

A genetic reversion analysis at the HIS4 locus in Saccharomyces cerevisiae has identified SUI1 as a component of the translation initiation complex which plays an important role in ribosomal recognition of the initiator codon. SUI1 is an essential protein of 12.3 kDa that is required in vivo for the initiation of protein synthesis. Here we present evidence that SUI1 is identical to the smallest subunit, p16, of eukaryotic translation initiation factor 3 (eIF-3) in S. cerevisiae. SUI1 and eIF3-p16 comigrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cross-react with anti-SUI1 and anti-eIF3 antisera. Anti-SUI1 antisera immunoprecipitate all of the subunits of eIF3, whereas antisera against the eIF3 complex and the individual PRT1 and GCD10 subunits of eIF3 immunoprecipitate SUI1. Finally, the N-terminal amino acid sequence of a truncated form of eIF3-p16 matches the sequence of SUI1. eIF3 isolated from a sui1(ts) strain at 37 degrees C lacks SUI1 and fails to exhibit eIF3 activity in the in vitro assay for methionyl-puromycin synthesis. A free form of SUI1 separate from the eIF3 complex is found in S. cerevisiae but lacks activity in the in vitro assay. The results, together with prior genetic experiments, indicate that SUI1 is essential for eIF3 activity and functions as part of eIF3 and in concert with eIF2 to promote eIF2-GTP-Met-tRNAi ternary complex recognition of the initiator codon.

Sign in / Sign up

Export Citation Format

Share Document