A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

Ana Garca-Sacrist an 1 2 Miguel Moreno 2 Ascensio n Ariza-Mateos 0 1 Elena Lo pez-Camacho 2 3 Rosa M. J audenes 2 Luis V azquez 3 Jordi Go mez 0 1 Jos e Angel Martn-Gago 2 3 Carlos Briones 1 2 0 Laboratory of RNA Archaeology, Instituto de Parasitolog a y Biomedicina 'L o pez-Neyra' (CSIC), Parque Tecnol o gico Ciencias de la Salud , Armilla, Granada 18016 , Spain 1 Centro de Investigaciones Biom e dicas en Red de Enfermedades Hep a ticas y Digestivas, (CIBERehd) , Spain 2 Department of Molecular Evolution, Centro de Astrobiolog a (CSIC-INTA), Torrej o n de Ardoz , Madrid 28850 , Spain 3 Instituto de Ciencia de Materiales de Madrid (CSIC) , Cantoblanco, Madrid 28049 , Spain 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 ntlong 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 longrange 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. - 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). The 5 UTR of HCV is highly conserved among all the viral genotypes and contains an internal ribosome entry site (IRES) element that drives cap-independent initiation of translation of the viral polyprotein (4,5). The minimum sequence required for HCV IRES activity spans nucleotides (nts) 39371 of the viral genome (6,7), and contains domains II to IV followed by the first 27 nts of the core coding sequence (4). Its secondary structure has been proposed using in silico RNA folding, covariation sequence analysis and biochemical methods (4,6,812) (Figure 1). The structures of individual HCV IRES domains or subdomains have been studied using techniques with atomic resolution (such as X-ray diffraction (XRD) and nuclear magnetic resonance (NMR)) (5,1316), while electron microscopy (EM) has been used to visualize the structure of HCV IRES in its free form (17) and bound to the ribosome (18). A model of the HCV IRES structure in solution, based on small-angle X-ray scattering (SAXS) in combination with molecular dynamics simulations, has been also published (19). HCV IRES is flanked upstream by a short stem-loop termed domain I, which is connected with domain II through a 23-nt-long ssRNA spacer region. Additionally, two downstream, structured domains termed V and VI (spanning nts 388510 of the viral genome and located within the core coding sequence (20)), are essential for HCV viability (21) through a still unknown RNA structure-based mechanism. Phylogenetic (22), functional (23) and structural (24) studies have shown that a 15-nt-long sequence at the I-II spacer region can anneal with a complementary sequence at the basal region of domain VI (Figure 1), thus promoting a closed conformation of the HCV IRES. This long-range RNA interaction is destabilized upon binding of the most abundant liver-specific microRNA, miR-122, which also interacts with the I-II spacer region by binding to two seed sites located 8 or 9 nts apart, depending on the viral isolate (25,26), and promotes the switch to an open IRES conformation (27). However, the transition from open to closed (or vice versa) HCV IRES conformations has not been described in the absence of any effector RNA or protein molecule. At the functional level, miR-122 binding has been associated to several effects including the increase of HCV RNA stability (28,29), reduction of the 5 decay rates of the viral genome in infected cells (30) and stimulation of IRESmediated translation (31). However, the distinct effects of miR-122 on translation observed in vitro and in cell culture (32,33) make it difficult to associate different HCV IRES conformations with miR-122 activities and to assess the role of miR-122 in the viral life cycle (34,35). This encourages the acquisition of additional structural and functional data on the interaction of miR-122 with HCV IRES in different experimental conditions. RNA folding depends on the concentration of cations around the negatively charged phosphate backbone. Due to its high charge density, Mg2+ ions stabilize RNA tertiary structure more efficiently than other divalent or monovalent cations (36), thus being essential for the functional folding of large RNAs (37,38). The Mg2+ concentration required for optimal HCV IRES activity in translation-competent extracts ranges from 1 to 2.5 mM (39). HCV IRES can promote translation initiation at 2.5 mM Mg2+ without requiring the complete set of initiation factors, and it is able to drive initiation factor-independent translation at 5 mM Mg2+ (40). Although the Mg2+-dependent secondary structure of the minimal HCV IRES (domains IIIV) has already been investigated (10), new technological approaches are needed to visualize the effect of Mg2+ on the tertiary fold of the HCV IRES in its natural sequence context. Atomic force microscopy (AFM) is a powerful nanotechnology-based tool for the structural analysis of a wide range of biological entities. It provides a 3D surface profile of the imaged sample without requiring any staining or coating, thus being less destructive and disruptive than EM. The nanometer resolution of this technique is optimal for the visualization of nucleic acid molecules (414 (...truncated)