scholarly journals Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

2008 ◽  
Vol 105 (40) ◽  
pp. 15293-15298 ◽  
Author(s):  
Alexander Litovchick ◽  
Jack W. Szostak
Keyword(s):  
Translation Initiation ◽  
Cyclic Peptide ◽  
Internal Ribosomal Entry Site ◽  
Treatment Modalities ◽  
Detectable Effect ◽  
Mrna Display ◽  
Entry Site ◽  
Positive Strand Rna ◽  
Hcv Ires

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >1013 different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 262-272 ◽  
Author(s):  
Rastislav Horos ◽  
Hanna IJspeert ◽  
Dagmar Pospisilova ◽  
Regine Sendtner ◽  
Charlotte Andrieu-Soler ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Fetal Liver ◽  
Internal Ribosomal Entry Site ◽  
Ribosomal Subunit ◽  
Translation Efficiency ◽  
Entry Site ◽  
Diamond Blackfan Anemia ◽  
Ribosomal Entry

Abstract Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

scholarly journals Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

2001 ◽  
Vol 75 (8) ◽  
pp. 3841-3850 ◽  
Author(s):  
Natalya L. Teterina ◽  
Denise Egger ◽  
Kurt Bienz ◽  
David M. Brown ◽  
Bert L. Semler ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Viral Proteins ◽  
Encephalomyocarditis Virus ◽  
Minus Strand ◽  
Entry Site ◽  
Sequence Production ◽  
Positive Strand Rna

ABSTRACT HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.

scholarly journals Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA

1999 ◽  
Vol 80 (9) ◽  
pp. 2319-2327 ◽  
Author(s):  
Takeshi Isoyama ◽  
Nobuhiko Kamoshita ◽  
Kotaro Yasui ◽  
Atsushi Iwai ◽  
Kazuko Shiroki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosomal Entry Site ◽  
Translation System ◽  
Hcv Rna ◽  
Entry Site ◽  
La Protein ◽  
Reticulocyte Lysates ◽  
Hcv Ires

Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES). Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures. To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeast Saccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation. In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA. However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting. I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation. These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES. Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection. The data are compatible with the low requirement of La protein for HCV IRES activity.

scholarly journals Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus

2001 ◽  
Vol 75 (8) ◽  
pp. 3719-3730 ◽  
Author(s):  
Wei Dong Zhao ◽  
Eckard Wimmer
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosomal Entry Site ◽  
Genome Replication ◽  
Entry Site ◽  
Internal Ribosomal Entry Sites ◽  
Ribosomal Entry ◽  
Hcv Ires

ABSTRACT Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging toFlaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5′ deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5′ border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Δcore-R-Luc-[PV]IRES-F-Luc-P2,3-3′NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.

scholarly journals A Unique 5′ Translation Element Discovered in Triticum Mosaic Virus

2015 ◽  
Vol 89 (24) ◽  
pp. 12427-12440 ◽  
Author(s):  
Robyn Roberts ◽  
Jincan Zhang ◽  
Laura K. Mayberry ◽  
Satyanarayana Tatineni ◽  
Karen S. Browning ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Translation Initiation ◽  
Plant Virus ◽  
Plant Viruses ◽  
Leader Sequence ◽  
Content Type ◽  
Internal Translation ◽  
Positive Strand Rna ◽  
Independent Translation

ABSTRACTSeveral plant viruses encode elements at the 5′ end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5′ m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to thePotyviridaefamily of positive-strand RNA viruses. We demonstrate that the TriMV 5′ leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5′ leader to a 300-nucleotide sequence that drives cap-independent translation from the 5′ end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5′ untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5′ UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5′ end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation.IMPORTANCEMany members of thePotyviridaefamily rely on their 5′ end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5′ leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5′ UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5′ UTR is capable of driving cap-independent translationin vitroandin vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNAin vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

scholarly journals Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

2020 ◽  
Vol 295 (7) ◽  
pp. 1843-1856
Author(s):  
Baptiste Panthu ◽  
Solène Denolly ◽  
Cendrine Faivre-Moskalenko ◽  
Théophile Ohlmann ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hcv Rna ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Sites ◽  
Subunit E ◽  
Cellular Mrnas ◽  
Hcv Ires

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5′UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

scholarly journals Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138

2010 ◽  
Vol 84 (18) ◽  
pp. 9472-9486 ◽  
Author(s):  
Lora Grainger ◽  
Louis Cicchini ◽  
Michael Rak ◽  
Alex Petrucelli ◽  
Kerry D. Fitzgerald ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Translation Initiation ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Reporter System ◽  
Entry Site ◽  
Sequence Element ◽  
Low Passage

ABSTRACT We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3′ coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3′ end of each transcript and is preceded by an extensive 5′ sequence (∼0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5′ ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5′ of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

scholarly journals Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation

2005 ◽  
Vol 86 (4) ◽  
pp. 1015-1025 ◽  
Author(s):  
Katerina I. Kalliampakou ◽  
Maria Kalamvoki ◽  
Penelope Mavromara
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Plasmid Vector ◽  
Firefly Luciferase ◽  
Detectable Effect ◽  
Dependent Manner ◽  
Entry Site ◽  
Upstream Gene ◽  
Ns5a Protein ◽  
Hcv Ires

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5′ non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.

scholarly journals Factor requirements for translation initiation on the Simian picornavirus internal ribosomal entry site

RNA ◽  
2007 ◽  
Vol 14 (2) ◽  
pp. 367-380 ◽  
Author(s):  
S. de Breyne ◽  
Y. Yu ◽  
T. V. Pestova ◽  
C. U.T. Hellen
Keyword(s):  
Translation Initiation ◽  
Internal Ribosomal Entry Site ◽  
Entry Site ◽  
Ribosomal Entry

scholarly journals Cancer-associated mRNAs regulated by the Helix-Loop-Helix motif of human EIF3A

2018 ◽  
Author(s):  
Marina Volegova ◽  
Jamie H.D. Cate
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Binding Motif ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Helix Loop Helix ◽  
Small Set

AbstractImproper regulation of translation initiation, a vital check-point of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) has been associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit in eIF3 interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiationin vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, includingMYC. We also demonstrate that the HLH motif of EIF3A acts specifically on the 5’-UTR ofMYCmRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.SummaryThe Helix Loop Helix motif of EIF3A controls translation of a small set of oncogenic cellular transcripts, includingMYC, and modulates the function of translation initiation factor EIF4A1 during translation initiation on select mRNAs.

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