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.

Keywords: Hepatitis C virus; RNA; SPR; interactions; kinetics; kissing loop.

FIGURE 1.

(A) Scheme of the HCV genome. The internal ribosome entry site (IRES) is on the 5′ side. The SL2 stem–loop is located in the 3′ UTR region. Seq9110 and 5BSL3.2 are located in the region coding for the NS5B protein. The region coding for the core protein (Core) is also shown. (B) Sequences and secondary structures of the RNAs. 5BSL3.2 was modified with a biotin tag at the 3′ end, allowing immobilization by streptavidin-biotin coupling on the sensor chips. The sequence in bold black indicates bases that could form Watson-Crick base pairs with SL2 (also in bold black). The sequence in bold gray indicates bases that could interact with Seq9110 or AS₉₁₁₀ (also in bold gray). Mini5BSL3.2 is a truncated version (double arrow) of 5BSL3.2 corresponding to the perfect stem–loop above the internal loop. AS₉₁₁₀ is an antisense oligonucleotide derivatized from Seq9110. For SL2, the sequences in italic gray indicate the palindromic sequence of SL2. Underlined bases for SL2 and Seq9110 indicate nucleotides that were replaced to generate hairpins of decreased affinity for 5BSL3.2: UGU was replaced by ACA for SL2, and GGG by AAA for Seq9110. SL2_WT1 and SL2_WT2 are two predicted secondary structures of SL2 obtained from the Mfold web server (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form). Bases in bold gray for SL2_kistem (A and C) indicate modifications that were made to convert SL2_WT2 in a perfect stem–loop structure. SL2_GC was designed to generate a hairpin of increased stability compared with SL2 without altering the palindromic sequence. SL2_AAU is a mutated version of SL2_GC in which CUA was mutated in AAU (in bold gray).

FIGURE 2.

Kinetic analysis by the SCK method of SL2 hairpins binding to 5BSL3.2. The experiments were performed at 10°C on a SAHC200m sensor chip, at a flow rate of 50 µL/min. Thirty to 70 RU of biotinylated 5BSL3.2 were immobilized on the sensor chip. The samples were injected sequentially in the order of increasing concentrations, 62.5 nM (first arrow from the left), 250 nM (second arrow), and 1000 nM (third arrow). The regeneration was achieved with a 2-min pulse of a mixture of 40% formamide, 3.6 M urea, and 30 mM EDTA prepared in milli-Q water. The gray curves represent the experimental data (two overlaid sensorgrams). The black line represents the fit of one sensorgram to a Langmuir 1:1 model according to Equations (1) and (2) reported in the Supplemental Material. (A) SL2 wild type (SL2_WT) and the mutated SL2 stem–loop (SL2aca) in which the central UGU sequence of the loop was replaced by ACA. (B) SL2_kistem.

FIGURE 3.

Native acrylamide gels of SL2 hairpins. Two µg of SL2_WT, SL2_kistem, SL2_GC, and SL2_AAU were prepared and were loaded on 15% (w/v) 75:1 acrylamide/bis(acrylamide) native gels as described in Materials and Methods. The gels were stained by “Stains-all.”

FIGURE 4.

Kinetic analysis of Seq9110 and AS₉₁₁₀ binding to 5BSL3.2. The sensor chip was prepared as described in Figure 2 and in the Materials and Methods. RNA samples were injected over the functionalized surface in duplicate. Two overlaid sensorgrams (gray curves) are shown for each injected concentration. The regeneration of the surface was achieved with a 2-min pulse of a mixture of 40% formamide, 3.6 M urea, and 30 mM EDTA prepared in milli-Q water. The black lines represent the fit of one set of sensorgrams by global fitting analysis to a Langmuir 1:1 model. (A) Injection of Seq9110. (B) Injection of AS9110.

FIGURE 5.

Kinetic analysis by the SCKODS method of SL2_kistem hairpin and Seq9110 binding to 5BSL3.2. The sensor chip was prepared as described in Figure 2 and in Materials and Methods. Seq9110 was first injected over the 5BSL3.2-coated surface at 1 µM (first arrow from the left). During the dissociation phase of the formed Seq9110-5BSL3.2 complex, SL2_kistem was injected sequentially in the order of increasing concentrations, 62.5 nM (second arrow), 250 nM (third arrow), and 1000 nM (fourth arrow). Experiments were performed with injections of Seq9110 only (referred to as Seq9110 alone). The regeneration of the surface was achieved with a 2-min pulse of a mixture of 40% formamide, 3.6 M urea, and 30 mM EDTA prepared in milli-Q water. The gray curves represent the experimental data (two overlaid sensorgrams for SL2_kistem and Seq9110 alone). The black line for Seq9110 alone represents the fit of the dissociation phase according to Equation (2). The black lines for SL2_kistem represent the fit to a Langmuir 1:1 model of interaction according to Equations (1) and (2) by the SCKODS method.

FIGURE 6.

Kinetic analysis of SL2 binding to itself. SL2_WT was biotinylated at its 5′ end and immobilized on a streptavidin-coated CM5 sensor chip. SL2 hairpins were injected in duplicate at 500 nM as indicated by the arrows. The experiments were performed at 10°C and at a flow rate of 50 µL/min. The truncated version of 5BSL3.2, mini5BSL3.2, was also injected over the SL2 functionalized surface.