Effect of Magnesium Ions on the Tertiary Structure of the Hepatitis C Virus IRES and Its Affinity for the Cyclic Peptide Antibiotic Viomycin

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Abstract
A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg2+ ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with Kd values of 25−55 μM. Interestingly, viomycin binding to the two fragments is affected differently by Mg2+; noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg2+-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg2+ ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.