Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

Barría, María Inés; González, Ángel F.; Vera-Otárola, Jorge; León, Ursula; Vollrath, Valeska; Marsac, Delphine; Monasterio, Octavio; Pérez-Acle, Tomás; Soza, Alejandro; López-Lastra, Marcelo

doi:10.1093/nar/gkn1022

Cited by 51 publications

(87 citation statements)

References 65 publications

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“…These regions are highly conserved among hepacivirus and related flaviviruses (Jubin et al 2000;Tuplin et al 2002;You et al 2004;Barria et al 2008), supporting the idea that this contact may also occur in other closely related viral systems. Interestingly, small changes in the degradation pattern of the apical loop IIIc and the region surrounding nucleotide 360 were observed in 59 end probing in the presence of the 39 construct.…”

Section: Discussionsupporting

confidence: 52%

“…Domain IIId acts as the main anchoring site for the 40S ribosomal subunit (Kolupaeva et al 2000;Lukavsky et al 2000;Babaylova et al 2009). The high sequence and structural conservation rate among the hepaciviruses and even related RNA viruses firmly supports its requirement for viral persistence (Jubin et al 2000;Barria et al 2008).…”

Section: Introductionmentioning

confidence: 85%

“…(B) Diagram of the transcripts encompassing different functional domains of both the 59 and the 39 ends of the HCV genome used in this study. (Barria et al 2008), is composed of two short helices separated by a loop E and capped by an apical loop folded into a U-turn motif ( Fig. 1A; Jubin et al 2000;Klinck et al 2000).…”

Section: Introductionmentioning

confidence: 99%

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A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

Romero-López¹,

Berzal‐Herranz²

2009

RNA

112

146

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The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 59 UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 39 UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 39 end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 39 end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA-RNA interaction between the 59 and 39 ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 59-39 end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.

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Section: Discussionsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 85%

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A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

Romero-López¹,

Berzal‐Herranz²

2009

RNA

112

146

View full text Add to dashboard Cite

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“…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).…”

mentioning

confidence: 80%

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.

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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 ...

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“…Further studies are needed to account for this relationship. Although the HCV IRES sequence is highly conserved, some mutations have been found to decrease translation efficiency to various extents (20). Naturally occurring sequence variations in HCV IRES could lead to changes in its activity (21), and the activities of IRES in HCV strains isolated from patients receiving IFN-a/ribavirin therapy varied from each other (22,23).…”

Section: Original Articlementioning

confidence: 99%

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

et al. 2011

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The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5 0 -end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs. ' 2011 International Society for Advancement of Cytometry Key terms hepatitis C virus; internal ribosome entry site; DsRed2; Azami Green; flow cytometry HEPATITIS C virus (HCV) is a major causative agent of liver cirrhosis and hepatocellular carcinoma (1). The HCV genome is a plus-strand RNA consisting of $9,600 nucleotides (nt) and has a single long open reading frame encoding a polyprotein of about 3,000 amino acids (2). Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5-7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.The HCV IRES activity has been studied, by using biochemical methods with luciferase genes as reporters (8-10), which have some flaws resulting from using cell lysates. In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid. To

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Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

Cited by 51 publications

References 65 publications

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

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

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

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