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Abstract
The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.
Highlights
Hepatitis C virus (HCV) is the major etiological agent of chronic liver disease
The Atomic force microscopy (AFM) analysis of the internal ribosome entry site (IRES)-574 in 80% formamide (Supplementary Figure S2) evidenced that part of its secondary structure is highly stable under harsh denaturing conditions, since the imaged molecules showed 65–80% of the theoretical molecular length
The height range of the imaged IRES-574 molecule in formamide might correspond to dsRNA regions lying on the surface, together with other elements of non-denatured secondary structure in different orientations
Summary
Hepatitis C virus (HCV) is the major etiological agent of chronic liver disease. There is no HCV vaccine and the traditional treatment based on a combination of alphainterferon (IFN) and ribavirin (RBV) failed in about half of the patients. The need for new, alternative therapeutic approaches has encouraged the exploration of HCV life cycle as well as the development of direct-acting antiviral agents that have substantially increased sustained virologic response, what suggests that IFN-free regimens could lead to HCV eradication [1]. With this aim, a thorough study of the native structure of the HCV genomic RNA is currently required, since certain structural/functional RNA elements, in particular those present at the 5 and 3 untranslated regions (UTR), are promising targets for antiviral therapy [2,3].
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