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 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 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 Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site

2001 ◽  
Vol 82 (4) ◽  
pp. 757-763 ◽  
Author(s):  
Lanja Saleh ◽  
René C. Rust ◽  
Ralf Füllkrug ◽  
Ewald Beck ◽  
Gergis Bassili ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Foot And Mouth Disease ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Mouth Disease Virus ◽  
Ires Element

In the life-cycle of picornaviruses, the synthesis of the viral polyprotein is initiated cap-independently at the internal ribosome entry site (IRES) far downstream from the 5′ end of the viral plus-strand RNA. The cis-acting IRES RNA elements serve as binding sites for translation initiation factors that guide the ribosomes to an internal site of the viral RNA. In this study, we show that the eukaryotic translation initiation factor eIF4G interacts directly with the IRES of foot-and-mouth disease virus (FMDV). eIF4G binds mainly to the large Y-shaped stem–loop 4 RNA structure in the 3′ region of the FMDV IRES element, whereas stem–loop 5 contributes only slightly to eIF4G binding. Two subdomains of stem–loop 4 are absolutely essential for eIF4G binding, whereas another subdomain contributes to a lesser extent to binding of eIF4G. At the functional level, the translational activity of stem–loop 4 subdomain mutants correlates with the efficiency of binding of eIF4G in the UV cross-link assay. This indicates that the interaction of eIF4G with the IRES is crucial for the initiation of FMDV translation. A model for the interaction of initiation factors with the IRES element is discussed.

scholarly journals A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

2000 ◽  
Vol 20 (2) ◽  
pp. 496-506 ◽  
Author(s):  
Sivan Henis-Korenblit ◽  
Naomi Levy Strumpf ◽  
Dan Goldstaub ◽  
Adi Kimchi
Keyword(s):  
Internal Ribosome Entry Site ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Apoptotic Cells ◽  
Entry Site ◽  
Translation Rate ◽  
Internal Ribosome Entry ◽  
Caspase Cleavage ◽  
Induced Apoptosis

ABSTRACT Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5′ untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

scholarly journals Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

1999 ◽  
Vol 73 (7) ◽  
pp. 6111-6113 ◽  
Author(s):  
René C. Rust ◽  
Kerstin Ochs ◽  
Karsten Meyer ◽  
Ewald Beck ◽  
Michael Niepmann
Keyword(s):  
Disease Virus ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Foot And Mouth Disease ◽  
Initiation Factor ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Mouth Disease Virus

ABSTRACT Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.

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 The Rhopalosiphum padi virus 5′ internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system

2004 ◽  
Vol 85 (6) ◽  
pp. 1565-1569 ◽  
Author(s):  
Elizabeth Royall ◽  
Kathryn E. Woolaway ◽  
Jens Schacherl ◽  
Stefan Kubick ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Internal Ribosome Entry Site ◽  
Expression System ◽  
Rhopalosiphum Padi ◽  
Baculovirus Expression System ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Baculovirus Expression ◽  
Initiation Of Translation

Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5′ UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.

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