Nuclear Magnetic Resonance Study of RNA Structures at the 3′-End of the Hepatitis C Virus Genome

Abstract

The 3′-end of the genomic RNA of the hepatitis C virus (HCV) embeds conserved elements that regulate viral RNA synthesis and protein translation by mechanisms that have yet to be elucidated. Previous studies with oligo-RNA fragments have led to multiple, mutually exclusive secondary structure predictions, indicating that HCV RNA structure may be context-dependent. Here we employed a nuclear magnetic resonance (NMR) approach that involves long-range adenosine interaction detection, coupled with site-specific 2H labeling, to probe the structure of the intact 3′-end of the HCV genome (385 nucleotides). Our data reveal that the 3′-end exists as an equilibrium mixture of two conformations: an open conformation in which the 98 nucleotides of the 3′-tail (3′X) form a two-stem–loop structure with the kissing-loop residues sequestered and a closed conformation in which the 3′X rearranges its structure and forms a long-range kissing-loop interaction with an upstream cis-acting element 5BSL3.2. The long-range kissing species is favored under high-Mg2+ conditions, and the intervening sequences do not affect the equilibrium as their secondary structures remain unchanged. The open and closed conformations are consistent with the reported function regulation of viral RNA synthesis and protein translation, respectively. Our NMR detection of these RNA conformations and the structural equilibrium in the 3′-end of the HCV genome support its roles in coordinating various steps of HCV replication.

Structure and function analysis of the essential 3′X domain of hepatitis C virus

“…NMR spectroscopy and small angle X-ray scattering (SAXS) experiments have been used to study the solution structure of domain 3 ′ X, both in isolation (Cantero-Camacho and Gallego 2015;Cantero-Camacho et al 2017;Kranawetter et al 2017) and complexed with 5BSL3.2 (Cantero-Camacho and Gallego 2018). When isolated, the domain adopts a conformation comprising two stem-loops, SL1 ′ and SL2 ′ , stabilized by coaxial stacking (Fig.…”

“…When isolated, the domain adopts a conformation comprising two stem-loops, SL1 ′ and SL2 ′ , stabilized by coaxial stacking (Fig. 1B; Cantero-Camacho and Gallego 2015;Cantero-Camacho et al 2017;Kranawetter et al 2017). This fold exposes a palindromic tetranucleotide sequence-the four central nt of the DLS-in the apical loop of subdomain SL2 ′ , promoting dimerization of the 3 ′ X domain at higher ionic strength and/or RNA concentration (Cantero-Camacho and Gallego 2015;Cantero-Camacho et al 2017).…”

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

“…For example, a recent study on the effects of Mg 2þ in RNA folding showed that the high concentration of Mg 2þ in liver may play a critical role in promoting the formation of an RNA kissing complex, a structure essential for efficient hepatitis C viral replication. This result suggests that Mg 2þ -dependent structural equilibrium may be an adaptive property of the hepatitis C genomic RNA (97)(98)(99). Furthermore, the results from the current study, including the analytical formulas for the dehydration free energy, may serve as a useful starting point for a systematic development of the dehydration free energy as a function of the structure environment of the binding site.…”

Challenges and approaches to predicting RNA with multiple functional structures

“…The final 98 nt of the 3 ′ end, also called 3X ′ , can form a structure with three hairpins or two hairpins, in which two hairpins have completely refolded to form one new longer hairpin. The presence of the kissing loop hairpin partner can induce this structural change (Kranawetter et al 2017). The 3X ′ sequence can also form a dimer that facilitates genome dimerization for packaging (Cantero-Camacho et al 2017).…”

“…NMR spectroscopy has been used to characterize these conformational changes. Selective deuterium labeling and lr-AID (Keane et al 2016), a method that identifies the unusual chemical shift of the central adenine H2 proton in the motif 5 ′ UAA3 ′ /3 ′ AUU5 ′ , has been used to characterize both the transition from open to kissing loop conformation and the structural rearrangements from two to three hairpins in 3X ′ (Kranawetter et al 2017). In addition, the Monte Carlo Tightly Bound Ion (MCTBI) and Vfold 3D RNA folding software have been used to model the different conformations of the 3 ′ -UTR and the metal ion binding sites.…”