scholarly journals Characterization of the 5′ internal ribosome entry site of Plautia stali intestine virus

2006 ◽  
Vol 87 (12) ◽  
pp. 3679-3686 ◽  
Author(s):  
Norihiro Shibuya ◽  
Nobuhiko Nakashima
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rhopalosiphum Padi ◽  
Open Reading Frames ◽  
Translation System ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Free Translation ◽  
Reticulocyte Lysate ◽  
Plautia Stali ◽  
Translation Systems

The RNA genome of Plautia stali intestine virus (PSIV; Cripavirus, Dicistroviridae) contains two open reading frames, the first of which is preceded by a 570 nt untranslated region (5′ UTR). The 5′ UTR was confirmed to be an internal ribosome entry site (IRES) using an insect cell lysate translation system: translation of a second cistron increased 14-fold in the presence of the 5′ UTR and a cap analogue did not inhibit translation of the second cistron. Deletion analysis showed that 349 bases corresponding to nt 225–573 in the PSIV genome were necessary for internal initiation. The PSIV 5′ IRES did not function in rabbit reticulocyte lysate or wheatgerm translation systems; however, the intergenic IRES for capsid translation of PSIV was functional in both systems, indicating that the 5′ IRES and the intergenic IRES have distinct requirements for their activities. Chemical and enzymic analyses of the 5′ IRES of PSIV indicate that its structure is distinct from that of Rhopalosiphum padi virus. Because 5′ IRES elements in some dicistroviruses have been reported to be active in plant and mammalian cell-free translation systems, there appears to be variation among dicistroviruses in the mechanism of translation initiation mediated by 5′ IRES elements.

scholarly journals The 5′ Untranslated Region of Rhopalosiphum padi Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems

2001 ◽  
Vol 75 (21) ◽  
pp. 10244-10249 ◽  
Author(s):  
Kathryn E. Woolaway ◽  
Konstantinos Lazaridis ◽  
Graham J. Belsham ◽  
Michael J. Carter ◽  
Lisa O. Roberts
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rhopalosiphum Padi ◽  
Encephalomyocarditis Virus ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Translation Systems

ABSTRACT Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5′ UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5′ UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, andDrosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5′ IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.

scholarly journals Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems

2007 ◽  
Vol 88 (5) ◽  
pp. 1583-1588 ◽  
Author(s):  
Elisabetta Groppelli ◽  
Graham J. Belsham ◽  
Lisa O. Roberts
Keyword(s):  
Untranslated Region ◽  
Rhopalosiphum Padi ◽  
Open Reading Frames ◽  
Distinct Region ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Initiation ◽  
Minimal Sequences ◽  
Translation Systems

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.

scholarly journals eIF4G-driven translation initiation of downstream ORFs in mammalian cells

2020 ◽  
Vol 48 (18) ◽  
pp. 10441-10455
Author(s):  
Risa Nobuta ◽  
Kodai Machida ◽  
Misaki Sato ◽  
Satoshi Hashimoto ◽  
Yasuhito Toriumi ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Translation System ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Genome Wide

Abstract Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3′ untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3′ UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3′ UTRs. The 3′ UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3′ UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3′ UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3′ UTRs of mRNAs may encode various products.

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.

scholarly journals Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

2015 ◽  
Vol 112 (47) ◽  
pp. E6446-E6455 ◽  
Author(s):  
Hilda H. Au ◽  
Gabriel Cornilescu ◽  
Kathryn D. Mouzakis ◽  
Qian Ren ◽  
Jordan E. Burke ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Structure ◽  
Hybrid Approach ◽  
Translation System ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
The Core ◽  
X Ray Scattering ◽  
Intermolecular Contacts

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirusIsraeli acute paralysis virus(IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

scholarly journals A Cross-Kingdom Internal Ribosome Entry Site Reveals a Simplified Mode of Internal Ribosome Entry

2005 ◽  
Vol 25 (17) ◽  
pp. 7879-7888 ◽  
Author(s):  
Ilya M. Terenin ◽  
Sergei E. Dmitriev ◽  
Dmitri E. Andreev ◽  
Elizabeth Royall ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Specific Binding ◽  
Rhopalosiphum Padi ◽  
In Vitro Translation ◽  
Maximum Efficiency ◽  
Marginal Effect ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors

ABSTRACT Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5′ untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with ∼23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5′-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

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