Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES

International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.
Nucleic Acids Research (Impact Factor: 9.11). 03/2000; 28(4):875-85.
Source: PubMed


Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

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Available from: Emanuele Buratti
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    • "This apparently simple process is guided by a subset of regulatory structural RNA elements included in the IRES element. Thus, the conformation of the IRES plays a crucial role in viral protein synthesis (14–18). "
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    ABSTRACT: Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3′-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3′-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3′-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA–RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.
    Full-text · Article · Oct 2012 · Nucleic Acids Research
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    • "This represents an alternative mechanism to that employed by cellular mRNAs, in which 40S ribosomal subunits are directly recruited in the absence of any other canonical initiation factor, directly positioning the start codon at the ribosome P site (Lytle et al. 2002; Ji et al. 2004; Otto and Puglisi 2004). The HCV IRES spans a region of 340 nucleotides (nt) that includes a short stretch of the 59 core coding sequence (Fig. 1A; Reynolds et al. 1995; Wang et al. 2000), and has a complex organization that must be preserved for it to be active (Lukavsky et al. 2000; Odreman-Macchioli et al. 2000; Collier et al. 2002; Kieft et al. 2002). Under physiological magnesium concentrations, the IRES is folded into four stem–loop motifs (designated I–IV) that define different functional domains (Fig. 1A), each with essential roles in ribosome recruitment and viral RNA synthesis. "
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    ABSTRACT: 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 5' UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3' 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 3' end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3' 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 5' and 3' 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 5'-3' end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.
    Full-text · Article · Aug 2009 · RNA
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    • "The respective contribution of the RNA hydrolysis versus the trigger of an inactive conformation of the IRES in the overall translation inhibition observed in vitro is unknown. Both the sequence and structure of domain III from the IRES are essential for the binding of eIF3 (3,16,41,42) and of ribosomal protein S9 (43). Interestingly, this is particularly true for domains IIIb and IIId (44), which are both cleaved by conjugate 1. "
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    ABSTRACT: Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.
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