scholarly journals Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

1996 ◽  
Vol 16 (12) ◽  
pp. 6859-6869 ◽  
Author(s):  
T V Pestova ◽  
C U Hellen ◽  
I N Shatsky
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Entry Site ◽  
Independent Manner ◽  
Initiation Factors ◽  
Virus Rna ◽  
Initiation Of Translation ◽  
Initiation Factor 2 ◽  
Ribosomal Entry

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

scholarly journals Physical Association of Eukaryotic Initiation Factor 4G (eIF4G) with eIF4A Strongly Enhances Binding of eIF4G to the Internal Ribosomal Entry Site of Encephalomyocarditis Virus and Is Required for Internal Initiation of Translation

2000 ◽  
Vol 20 (16) ◽  
pp. 6019-6029 ◽  
Author(s):  
Ivan B. Lomakin ◽  
Christopher U. T. Hellen ◽  
Tatyana V. Pestova
Keyword(s):  
Complex Formation ◽  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Rna Recognition Motif ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Amino Terminal ◽  
Initiation Of Translation ◽  
Ribosomal Entry

ABSTRACT Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and β-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not β-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.

scholarly journals Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

2003 ◽  
Vol 23 (2) ◽  
pp. 687-698 ◽  
Author(s):  
Victoria G. Kolupaeva ◽  
Ivan B. Lomakin ◽  
Tatyana V. Pestova ◽  
Christopher U. T. Hellen
Keyword(s):  
Conformational Changes ◽  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Initiation Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Central Domain ◽  
C Terminus ◽  
Initiation Of Translation

ABSTRACT Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3′ border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

scholarly journals Promotion of viral internal ribosomal entry site-mediated translation under amino acid starvation

2012 ◽  
Vol 93 (5) ◽  
pp. 951-962 ◽  
Author(s):  
Maria Licursi ◽  
Yumiko Komatsu ◽  
Theerawat Pongnopparat ◽  
Kensuke Hirasawa
Keyword(s):  
Amino Acid ◽  
Internal Ribosomal Entry Site ◽  
Amino Acid Starvation ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Ribosomal Entry ◽  
Cellular Stresses

Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation.

scholarly journals Heat Shock Protein A6, a Novel HSP70, Is Induced During Enterovirus A71 Infection to Facilitate Internal Ribosomal Entry Site-Mediated Translation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu-Siang Su ◽  
Lih-Hwa Hwang ◽  
Chi-Ju Chen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Neurological Diseases ◽  
Internal Ribosomal Entry Site ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Enterovirus A71 ◽  
Ribosomal Entry

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot, and mouth disease (HFMD) in children. Its infection can lead to severe neurological diseases or even death in some cases. While being produced in a large quantity during infection, viral proteins often require the assistance from cellular chaperones for proper folding. In this study, we found that heat shock protein A6 (HSPA6), whose function in viral life cycle is scarcely studied, was induced and functioned as a positive regulator for EV-A71 infection. Depletion of HSPA6 led to the reductions of EV-A71 viral proteins, viral RNA and virions as a result of the downregulation of internal ribosomal entry site (IRES)-mediated translation. Unlike other HSP70 isoforms such as HSPA1, HSPA8, and HSPA9, which regulate all phases of the EV-A71 life, HSPA6 was required for the IRES-mediated translation only. Unexpectedly, the importance of HSPA6 in the IRES activity could be observed in the absence of viral proteins, suggesting that HSPA6 facilitated IRES activity through cellular factor(s) instead of viral proteins. Intriguingly, the knockdown of HSPA6 also caused the reduction of luciferase activity driven by the IRES from coxsackievirus A16, echovirus 9, encephalomyocarditis virus, or hepatitis C virus, supporting that HSPA6 may assist the function of a cellular protein generally required for viral IRES activities.

scholarly journals Structural characterization of ribosome recruitment and translocation by type IV IRES

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jason Murray ◽  
Christos G Savva ◽  
Byung-Sik Shin ◽  
Thomas E Dever ◽  
V Ramakrishnan ◽  
...  
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Post Translational Modification ◽  
Entry Site ◽  
Small Ribosomal Subunit ◽  
Initiation Factors ◽  
Type Iv ◽  
Peptidyl Transfer ◽  
Initiation Of Translation

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (<xref ref-type="bibr" rid="bib19">Fernández et al., 2014</xref>; <xref ref-type="bibr" rid="bib34">Koh et al., 2014</xref>), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

scholarly journals Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3353-3359 ◽  
Author(s):  
Oréda Boussadia ◽  
Michael Niepmann ◽  
Laurent Créancier ◽  
Anne-Catherine Prats ◽  
François Dautry ◽  
...  
Keyword(s):  
Rna Binding ◽  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

scholarly journals Conserved Nucleotides within the J Domain of the Encephalomyocarditis Virus Internal Ribosome Entry Site Are Required for Activity and for Interaction with eIF4G

2003 ◽  
Vol 77 (23) ◽  
pp. 12441-12449 ◽  
Author(s):  
Angela T. Clark ◽  
Morwenna E. M. Robertson ◽  
Graeme L. Conn ◽  
Graham J. Belsham
Keyword(s):  
Internal Ribosome Entry Site ◽  
Single Point ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation ◽  
Mouth Disease Virus ◽  
J Domain

ABSTRACT The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.

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