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.
    Nucleic Acids Research 10/2012; 40(22). DOI:10.1093/nar/gks927 · 9.11 Impact Factor
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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.
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    • "It is not clear whether these factors need a sequence motif or a RNA secondary structure/sequence motif combination to bind to IRES-containing mRNAs. In viral IRESes, like HCV and EMCV, RNA secondary structure has been shown to be crucial for IRES function (14–19). These structures are also conserved in other viruses that do not share primary sequence similarity. "
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    ABSTRACT: Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function.
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