scholarly journals In vitro and in vivo identification of structural and sequence elements in the 5′ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation

2006 ◽  
Vol 87 (12) ◽  
pp. 3667-3677 ◽  
Author(s):  
Jie Lu ◽  
Jiamin Zhang ◽  
Xiaochun Wang ◽  
Hong Jiang ◽  
Chuanfeng Liu ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Site Directed Mutagenesis ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Sequence Elements ◽  
Internal Initiation

Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5′ untranslated region (5′ UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5′ UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem–loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem–loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391–394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.

scholarly journals The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus

2001 ◽  
Vol 82 (9) ◽  
pp. 2257-2269 ◽  
Author(s):  
Tracey M. Hinton ◽  
Brendan S. Crabb
Keyword(s):  
Encephalomyocarditis Virus ◽  
Site Directed Mutagenesis ◽  
Type Ii ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Stem Loop ◽  
Mammalian Expression ◽  
B Virus

Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem–loop structures (H–L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189–920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem–loops H–L as well as four additional upstream stem–loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869–880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.

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 Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding

2000 ◽  
Vol 74 (22) ◽  
pp. 10430-10437 ◽  
Author(s):  
Ronald Jubin ◽  
Nicole E. Vantuno ◽  
Jeffrey S. Kieft ◽  
Michael G. Murray ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Apical Loop ◽  
Hcv Ires

ABSTRACT The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus andPestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T1-probing studies, demonstrating the importance of correct RNA folding to IRES function.

scholarly journals Interactions of Viral Protein 3CD and Poly(rC) Binding Protein with the 5′ Untranslated Region of the Poliovirus Genome

2000 ◽  
Vol 74 (5) ◽  
pp. 2219-2226 ◽  
Author(s):  
Andrea V. Gamarnik ◽  
Raul Andino
Keyword(s):  
Viral Protein ◽  
Untranslated Region ◽  
Rna Binding ◽  
Binding Protein ◽  
Cellular Protein ◽  
Site Directed Mutagenesis ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Cloverleaf Structure

ABSTRACT The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5′ untranslated region (5′UTR), the 5′ cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5′ cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5′UTR are modulated by the viral protein 3CD.

scholarly journals Mutational Analysis of the GB Virus B Internal Ribosome Entry Site

2000 ◽  
Vol 74 (2) ◽  
pp. 773-783 ◽  
Author(s):  
René Rijnbrand ◽  
Geoffrey Abell ◽  
Stanley M. Lemon
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mutational Analysis ◽  
General Structure ◽  
Point Mutations ◽  
Structural Domain ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry

ABSTRACT GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5′ nontranslated GBV-B RNA (5′NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5′NTR (M. Honda, et al. RNA 2:955–968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5′ end by structural domain II, a location analogous to the 5′ limit of the IRES in both the HCV and pestivirus 5′NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3′ limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

scholarly journals Production and Application of Multicistronic Constructs for Various Human Disease Therapies

Pharmaceutics ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 580 ◽  
Author(s):  
Alisa A. Shaimardanova ◽  
Kristina V. Kitaeva ◽  
Ilmira I. Abdrakhmanova ◽  
Vladislav M. Chernov ◽  
Catrin S. Rutland ◽  
...  
Keyword(s):  
Human Diseases ◽  
Cellular Biology ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
New Methods ◽  
In Vivo Experiments ◽  
Mutant Genes ◽  
Methods Of Treatment

The development of multicistronic vectors has opened up new opportunities to address the fundamental issues of molecular and cellular biology related to the need for the simultaneous delivery and joint expression of several genes. To date, the examples of the successful use of multicistronic vectors have been described for the development of new methods of treatment of various human diseases, including cardiovascular, oncological, metabolic, autoimmune, and neurodegenerative disorders. The safety and effectiveness of the joint delivery of therapeutic genes in multicistronic vectors based on the internal ribosome entry site (IRES) and self-cleaving 2A peptides have been shown in both in vitro and in vivo experiments as well as in clinical trials. Co-expression of several genes in one vector has also been used to create animal models of various inherited diseases which are caused by mutations in several genes. Multicistronic vectors provide expression of all mutant genes, which allows the most complete mimicking disease pathogenesis. This review comprehensively discusses multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides, including its features and possible application for the treatment and modeling of various human diseases.

scholarly journals Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated

2007 ◽  
Vol 27 (13) ◽  
pp. 4685-4697 ◽  
Author(s):  
Sergey E. Dmitriev ◽  
Dmitri E. Andreev ◽  
Ilya M. Terenin ◽  
Ivan A. Olovnikov ◽  
Vladimir S. Prassolov ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
High Efficiency ◽  
Rna Binding ◽  
Cultured Cells ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Mrnas ◽  
Internal Initiation

ABSTRACT Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4795-4795
Author(s):  
Patrick J. Frost ◽  
YiJiang Shi ◽  
Carolyne Bardalaban ◽  
Bao Hoang ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Mtor Inhibitors ◽  
G1 Arrest ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Myeloma Cells ◽  
Transfected Cells ◽  
P38 Inhibitor

Abstract In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT’s regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.

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