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

Jubin, Ronald; Vantuno, Nicole E.; Kieft, Jeffrey S.; Murray, Michael G.; Doudna, Jennifer A.; Lau, Johnson Y.N.; Baroudy, Bahige M.

doi:10.1128/jvi.74.22.10430-10437.2000

Cited by 96 publications

(95 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This IRES-rRNA interaction is supported by studies showing that mutations in the HCV IRES at nucleotides 266 GGG 268 , which are predicted to disrupt base pairing to 18S rRNA, drastically reduced the binding affinity of the IRES to 40S subunits (8,19). These mutations also disrupted IRES activity, both in vitro and in cells (19)(20)(21)(22)(23). In addition, when complexed with 40S subunits, the IIId loop of the HCV IRES was protected from cleavage by RNase T1 (8,24) or from modification by kethoxal (25).…”

supporting

confidence: 52%

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Matsuda

Mauro

2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example-translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the ShineDalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.hepatitis C virus | 18S rRNA | IRES | base pairing | translation H CV is a single-stranded RNA virus that is a major cause of severe liver disease. The RNA genome contains a large ORF and expresses a single polypeptide that is processed into smaller proteins, which are necessary for replication and assembly of viral particles. The RNA genome is uncapped, and translation does not require the eukaryotic initiation factor 4F (eIF4F) complex (1), which mediates cap-dependent translation. Instead, translation is facilitated by an internal ribosome entry site (IRES) located in the 5′ nontranslated region (2, 3). Inasmuch as the production of all HCV proteins requires the HCV IRES, the HCV IRES is a therapeutic target (4).IRESs encompass a variety of initiation mechanisms and have been extensively studied in viruses, which often exploit the translation capabilities of the host for their own use (5-7). In many cases, the IRES mechanism complements the infection strategy of the virus. In the case of the HCV IRES and other IRESs of this type, the IRES can effectively recruit the host translation machinery by directly binding to 40S ribosomal subunits (1,(8)(9)(10)(11)(12). The HCV-ribosome interaction has been shown to require an interaction with ribosomal protein S25 (13) and can also involve the eIF3 complex, which increases the stability of the interaction (14). In the presence of ternary complex, which contains the initiator Met-tRNA, a preinitiation complex can assemble at the start site.Various studies have investigated the binding of ...

show abstract

supporting

confidence: 52%

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Matsuda

Mauro

2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…However, neither of these deleterious deletions abrogated completely the IRES activity of Hsp70 mRNA. This contrasts with the effect of small deletions or even point mutations on viral IRESs, at least those characterized to data (21,(25)(26)(27)(28)(29)(30)(31)(32). Some of such mutations completely abolish their translation initiation activity (see, for example, the data for one of the EMCV IRES mutants in Fig.…”

Section: Ires Properties Of the 5ј-utr Of Hsp70 Mrna Are Not Due To Ementioning

confidence: 52%

“…These domains include highly specific binding sites for canonical initiation factors, auxiliary mRNA-binding proteins, or the 40 S ribosomal subunit (4). That is why short deletions or even point mutations in many parts of these IRESs are able to completely destroy them (21,(25)(26)(27)(28)(29)(30)(31)(32). This does not appear to be the case for the IRES of Hsp70 mRNA.…”

Section: Discussionmentioning

confidence: 96%

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

Rubtsova

Sizova

Dmitriev

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5 -untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5 -UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5 -UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5 -UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5 -terminal half of the 5 -UTR and rather strong for the 3 -terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5 -UTR from the 5 -end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5 -UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5 -UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

show abstract

“…Recently, a morpholino oligonucleotide complementary to start codon region of the HCV IRES was shown to reduce translation in rabbit reticulocyte lysates by greater than 80%. 7 To date, however, there have been few reports of morpholino efficacy in mammals. 18 A small animal model that uses a noninvasive assay for rapid and repeated measurements of HCV IRES inhibition would greatly accelerate the in vivo evaluation of morpholino oligonucleotides as therapeutic.…”

mentioning

confidence: 99%

“…The conserved HCV IRES is a likely target for effective therapeutic intervention. Extensive effort has been devoted to the development of antisense oligonucleotide, [1][2][3][4][5][6][7] ribozyme, [8][9][10] and DNA ribonuclease 11 inhibitors targeting HCV IRES sequences that are accessible for nucleic acid hybridization.…”

mentioning

confidence: 99%

A Potent and Specific Morpholino Antisense Inhibitor of Hepatitis C Translation in Mice

et al. 2003

View full text Add to dashboard Cite

C ap-independent translation of the hepatitis C virus (HCV) polyprotein initiates at a highly structured internal ribosome entry site (IRES) at the 5Ј end of the genomic RNA. The conserved HCV IRES is a likely target for effective therapeutic intervention. Extensive effort has been devoted to the development of antisense oligonucleotide, 1-7 ribozyme, [8][9][10] and DNA ribonuclease 11 inhibitors targeting HCV IRES sequences that are accessible for nucleic acid hybridization.Although in vitro assays have been invaluable in the early evaluation of inhibitors, work by our group 12 and that of others 13,14 suggests that cell-free systems may not faithfully recapitulate translation from the HCV IRES. A recent report that HCV IRES translation varies with cell cycling 15 also raises concern that rapidly dividing cells in tissue culture may not accurately model the normally quiescent human liver. Therefore, model systems that allow evaluation of pharmacokinetic parameters such as inhibitor bioavailability, toxicity, and systemic clearance in the complex milieu of a mammalian organism would be useful. Preclinical small animal models for testing nucleic acid inhibitors of HCV will help move candidate inhibitors into the clinic. Recently, a mouse xenotransplantation model was reported that supports HCV viral replication in transplanted human liver cells. 16 Although very exciting, this system requires access to fresh human liver tissue. A vaccinia virus expressing a HCV IRES luciferase fusion also has been used as a model system for evaluating antisense inhibitors in vivo. 4 In this model, a phosphorothioate and a 5-methylcytidine antisense oligonucleotide moderately reduced expression from the HCV IRES (ϳ50% and 80%). However, nonspecific effects

show abstract

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

Cited by 96 publications

References 40 publications

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

A Potent and Specific Morpholino Antisense Inhibitor of Hepatitis C Translation in Mice

Contact Info

Product

Resources

About