Article

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

ABSTRACT

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.
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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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    • "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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