scholarly journals Translation Eukaryotic Initiation Factor 4G Recognizes a Specific Structural Element within the Internal Ribosome Entry Site of Encephalomyocarditis Virus RNA

1998 ◽  
Vol 273 (29) ◽  
pp. 18599-18604 ◽  
Author(s):  
Victoria G. Kolupaeva ◽  
Tatyana V. Pestova ◽  
Christopher U. T. Hellen ◽  
Ivan N. Shatsky
Keyword(s):  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Structural Element ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Virus Rna ◽  
Eukaryotic Initiation Factor 4G

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.

scholarly journals Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent

2001 ◽  
Vol 75 (24) ◽  
pp. 12141-12152 ◽  
Author(s):  
Daniel R. Gallie
Keyword(s):  
Large Subunit ◽  
Tobacco Etch Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4G ◽  
Independent Translation

ABSTRACT The 5′ leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

scholarly journals Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2α

2004 ◽  
Vol 385 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Sandrine A. TINTON ◽  
Bert SCHEPENS ◽  
Yanik BRUYNOOGHE ◽  
Rudi BEYAERT ◽  
Sigrid CORNELIS
Keyword(s):  
Cell Cycle ◽  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Initiation Of Translation ◽  
Cycle Dependent

The PITSLRE kinases belong to the large family of cyclin-dependent protein kinases. Their function has been related to cell-cycle regulation, splicing and apoptosis. We have previously shown that the open reading frame of the p110PITSLRE transcript contains an IRES (internal ribosome entry site) that allows the expression of a smaller p58PITSLRE isoform during the G2/M stage of the cell cycle. In the present study we investigated further the role of cis- and trans-acting factors in the regulation of the PITSLRE IRES. Progressive deletion analysis showed that both a purine-rich sequence and a Unr (upstream of N-ras) consensus binding site are essential for PITSLRE IRES activity. In line with these observations, we demonstrate that the PITSLRE IRES interacts with the Unr protein, which is more prominently expressed at the G2/M stage of the cell cycle. We also show that phosphorylation of the α-subunit of the canonical initiation factor eIF-2 is increased at G2/M. Interestingly, phosphorylation of eIF-2α has a permissive effect on the efficiency of both the PITSLRE IRES and the ornithine decarboxylase IRES, two cell cycle-dependent IRESs, in mediating internal initiation of translation, whereas this was not observed with the viral EMCV (encephalomyocarditis virus) and HRV (human rhinovirus) IRESs.

scholarly journals Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

1996 ◽  
Vol 16 (12) ◽  
pp. 6870-6878 ◽  
Author(s):  
T V Pestova ◽  
I N Shatsky ◽  
C U Hellen
Keyword(s):  
Binding Protein ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Structural Element ◽  
Entry Site ◽  
Independent Manner ◽  
Central Domain ◽  
Nontranslated Region ◽  
Carboxy Terminal

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

scholarly journals Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct

2007 ◽  
Vol 81 (22) ◽  
pp. 12619-12629 ◽  
Author(s):  
Klaus K. Orlinger ◽  
Regina M. Kofler ◽  
Franz X. Heinz ◽  
Verena M. Hoenninger ◽  
Christian W. Mandl
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Replication ◽  
Particle Formation ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Expression Vectors ◽  
Nucleotide Substitutions ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3′ noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.

scholarly journals Phosphorylation of eIF2α is responsible for the failure of the picornavirus internal ribosome entry site to direct translation from Sindbis virus replicons

2013 ◽  
Vol 94 (4) ◽  
pp. 796-806 ◽  
Author(s):  
Miguel Angel Sanz ◽  
Natalia Redondo ◽  
Manuel García-Moreno ◽  
Luis Carrasco
Keyword(s):  
Internal Ribosome Entry Site ◽  
Sindbis Virus ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Wild Type ◽  
Entry Site ◽  
Embryonic Fibroblasts ◽  
Internal Ribosome Entry ◽  
Eif2α Phosphorylation ◽  
Wild Type Mefs

Translation directed by the poliovirus (PV) or encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) is very inefficient when expressed from Sindbis virus (SV) replicons. This inhibition can be rescued by co-expression of PV 2A protease (2Apro). Inhibition correlates with the extensive phosphorylation of eukaryotic initiation factor (eIF) 2α induced by SV replication. Confirmation that PV or EMCV IRES-driven translation can function when eIF2α is not phosphorylated was obtained in dsRNA-activated protein kinase knockout mouse embryonic fibroblasts (PKR−/− MEFs), where SV replication cannot induce eIF2α phosphorylation, and in variant S51A MEFs that express an unphosphorylatable eIF2α. In these cells, PV or EMCV IRES-dependent translation operated more efficiently than in wild-type MEFs. However, this translation was potently blocked when eIF2α was phosphorylated by the addition of thapsigargin to PKR−/− MEFs. In addition, when wild-type eIF2α was expressed in S51A MEFs or PKR was expressed in PKR−/− MEFs, PV IRES-dependent translation decreased. In both cases, the decrease in PV IRES-dependent translation correlated with the phosphorylation of eIF2α. Notably, PV 2Apro expression rescued PV IRES-driven translation in thapsigargin-treated PKR−/− MEFs. Taken together, these results demonstrated that PV IRES-driven translation can take place from SV replicons if eIF2α remains unphosphorylated. Remarkably, PV IRES-dependent translation was fully functional in this system when PV 2Apro was present, even if eIF2α was phosphorylated.

scholarly journals Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp

2005 ◽  
Vol 79 (2) ◽  
pp. 677-683 ◽  
Author(s):  
Randal C. Cevallos ◽  
Peter Sarnow
Keyword(s):  
Internal Ribosome Entry Site ◽  
Intergenic Region ◽  
Rna Virus ◽  
Penaeid Shrimp ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Functional Conservation

ABSTRACT The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

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