Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Abstract

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3 ′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

Predicting Ion Effects in an RNA Conformational Equilibrium

“…In agreement with the previous findings, 71, 72 the 5BSL3.2:3′X complex band can be detected only in gels containing Mg 2+ [see Fig. 5].…”

“…68–70 Previous gel electrophoresis and surface plasmon resonance (SPR) studies have shown that in vitro transcribed 5BSL3.2 and 3′X can form an RNA:RNA complex and Mg 2+ is required in the gel and running buffer in order to detect the RNA complex band. 71, 72 We apply the PCTBI model developed in the present study to investigate the physical mechanism for the Mg 2+ -dependence of the folding stability and the shift of conformational equilibrium for the 5BSL3.2:3′X kissing complex. Our results indicate that Mg 2+ ions can indeed stabilize the 5BSL3.2:3′X docked state over the undocked state and for 3′X, adding Mg 2+ to the solution shifts the conformational equilibrium toward the kissing conformation.…”

Structure of a Complex Formed by a Protein and a Helical Aromatic Oligoamide Foldamer at 2.1 Å Resolution

“…[23] Kinetic titration assays [24] were first performed with model compound 3 on HCA immobilized on the chip, and gave a K D value of approximately 5 nm (Figure 1). [23] Kinetic titration assays [24] were first performed with model compound 3 on HCA immobilized on the chip, and gave a K D value of approximately 5 nm (Figure 1).…”

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

“…25,30,32 The three-stem–loop (3SL) model (Figure 1C) is believed to be the dominant conformation because the kissing-loop residues in SL2 (nucleotides 9582–9588) are readily exposed and thus supports the long-range 5BSL3.2:3′X interaction. 11–13 The interaction between the SL2 and 5BSL3.2 RNA fragments has been observed by in vitro RNA:RNA binding assays, 23,33 but not by nuclear magnetic resonance (NMR) analysis. 11 Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and chemical probing studies showed that the nucleotide activities of 3′X residues were different in the presence of 5BSL3.2, 19,34 supporting the proposed 5BSL3.2:3′X interaction.…”