A magnesium-dependent RNA structural switch at the Internal Ribosome Entry Site of Hepatitis C Virus genome monitored by Atomic Force Microscopy

Abstract

Hepatitis C virus (HCV) is the main etiological agent of chronic liver disease in humans. Both 5 ́and 3 ́ untranslatable regions (UTR) of the single-stranded RNA HCV genome are highly structured and include regulatory elements necessary for viral replication and translation (1). In particular, the 5 ́UTR is highly conserved among all HCV genotypes and contains an internal ribosome entry site (IRES) element responsible to drive cap-independent translation initiation (2). The ion-dependent tertiary fold of the minimal HCV IRES element (containing domains II to IV) has been investigated (3), and significant progress has been made in determining the three-dimensional structure of individual IRES domains and subdomains at high resolution (4). Nevertheless, little information is still available on the tertiary structure of the whole functional HCV IRES element. Atomic Force Microscopy (AFM) is a useful nanotechnology-based tool for the analysis of a wide range of biological entities, including nucleic acids and their complexes (5). We have optimized AFM technology for analysing HCV IRES structure in native conditions as well as for monitoring its conformational changes in diverse physicochemical environments, in particular at magnesium ion concentrations ranging from 0 to 10 mM. Here we report the magnesium-dependent folding of the HCV IRES in a sequence context that includes its structured, functionally relevant flanking regions (domains I, V and VI). In the 568 nt-long HCV genomic RNA molecule analyzed, a structural switch has been monitored when magnesium concentration increases from 2 to 4 mM. This effect has been confirmed by classical techniques for RNA structural characterization such as gel-shift analysis and partial RNase T1 cleavage. Our results suggest a magnesium-driven transition from an ‘open’ to a ‘closed’ conformation of the HCV IRES, at least partially similar to that caused by miR-122 (6). The functional relevance of such an RNA structural switch will be discussed.