An unexpected RNA distal interaction mode found in an essential region of the hepatitis C virus genome

Figures

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  Figure 1.. RNA sequences and distal contacts involving the 3’X terminal domain of HCV. (A) Schematic representation of functional cis-acting structures identified in the 3′ region of the HCV genome. Distal RNA–RNA interactions proposed to have functional relevance are identified with double-headed arrows. (B) Predicted secondary structure of RNA sequences analyzed in this study: 98-nt full-length domain 3’X, 55-nt subdomain SL2’, 46-nt subdomain 5BSL3.2 (cre46) and 26-nt apical 5BSL3.2 hairpin cre26. 3’Xm and SL2’m are dimerization-defective variants of the wild-type sequences 3’X and SL2’, respectively, containing a double C29G/A31U mutation in the apical palindromic tetraloop of SL2’. SL2’m57 is a dimerization-defective subdomain SL2’ variant with two G:U pairs replaced with G:C and a G:C pair added in the lower stem (blue squares). The blue-colored nucleotides in cre26 and cre46 indicate changes relative to the wild-type sequence, introduced to increase transcription yield. 3’X sequence k (indicated with green circles) has been shown to establish a distal interaction involving Watson–Crick pairs with a complementary k’ sequence (green circles) in the apical loop of 5BSL3.2. In the…

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  Figure 2.. Gel electrophoresis analysis of 3’X domain sequences and their complexes with the 5BSL3.2 hairpin cre26. (A) Native gel comparing the electrophoretic mobility of wild-type 3’X and SL2’ and dimerization-defective 3’Xm and SL2’m constructs in the absence and presence of one molar equivalent of cre26. (B) Native gel comparing the electrophoretic mobility of constructs SL2’m and SL2’m57 in the absence and presence of one molar equivalent of cre26. All experiments used a running buffer containing Mg2+ ions, which have been shown to promote homodimerization and 5BSL3.2 complex formation.

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  Figure 3.. NMR spectroscopy analysis of the distal interaction formed between full-length domain sequence 3’Xm and 5BSL3.2 hairpin cre26. (A) Superposition of 1H-15N HNN-COSY spectra of 3’Xm, acquired in the absence (green) and presence (blue) of one molar equivalent of unlabeled cre26 sequence. Since the experiment used unlabeled cre26, all of the HN and HNN crosspeaks correspond to 3’Xm. The crosspeaks of upper SL2’ stem nucleotides are identified with green labels, and the HNN crosspeaks that disappear with the addition of cre26 are marked with green circles. The HN crosspeak that appears upon cre26 binding is marked in orange and was assigned as U34. Crosspeaks marked with crosses are visible at a lower contour level. The crosspeak corresponding to a minor conformation of U36 is marked with an asterisk. (B) Secondary structure of isolated domain 3’Xm supported by the NMR data. DLS nucleotides are depicted in red, k nucleotides are indicated with green circles, and the C29G and A31U mutations blocking dimerization are colored blue. Residues with assigned HNN resonances are numbered, and those whose HNN-COSY crosspeak patterns undergo changes upon addition of cre26 are marked with green…

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  Figure 4.. Schematic representation of the base-pairs proposed to be formed by the k nucleotides of the SL2’ subdomain in the absence (top) and presence (bottom) of 5BSL3.2 hairpin cre26. SL2’ DLS nucleotides are shown in red and marked with asterisks, and k and k’ residues are identified with green circles. The NH··N hydrogen bonds present in the base pairs are only detected by NMR HNN-COSY experiments when the two bases are 15N-labeled.

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  Figure 5.. NMR spectroscopy analysis of subdomain SL2’m in the absence and presence of 5BSL3.2 hairpin cre26. (A) Comparison of 1H-15N HNN-COSY spectra of construct SL2’m acquired in the absence (green) and presence (blue) of one molar equivalent of unlabeled cre26 sequence. Since the experiment used unlabeled cre26, all of the HN and HNN crosspeaks correspond to SL2’m. The crosspeaks of upper SL2’ stem nucleotides are identified with green labels, and the HNN crosspeaks that disappear with the addition of cre26 are marked with green circles. The HN diagonal crosspeak that appears upon cre26 binding is marked in orange and was assigned as U34. The C8 HNN crosspeak marked with a cross is visible at a lower contour level in the absence of cre26. The crosspeak corresponding to a minor conformation of U36 (see Supplementary Figure S2) is marked with an asterisk. (B) Superposition of selected regions of the 1H-13C HSQC spectra of construct SL2’m acquired in the absence (green) and presence of one (blue) and two (red) molar equivalents of unlabeled cre26 sequence. In the C2-H2 region (left), A25 H2 in the upper stem disappears when adding cre26, whereas A49 H2 (lower stem) remains relatively…

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  Figure 6.. NMR spectroscopy analysis of 5BSL3.2 hairpin cre26 in the absence and presence of subdomain SL2’m. (A) Superposition of selected regions of the 1H-13C HSQC spectra of construct cre26 acquired in the absence (green) and presence (blue) of one molar equivalent of unlabeled SL2’m sequence. k’ nucleotides are marked with green font. Note the strong shifts of k’ nucleotides C15’ and C18’ in the C5-H5 region (top), as well as A14’ and A16’ in the C2-H2 section (bottom). (B) NMR-supported secondary structure of isolated hairpin cre26, with k’ nucleotides indicated with green circles. Residues whose aromatic carbon and hydrogen resonances (C/H2, C/H5, C/H6 and/or C/H8) undergo significant chemical shift changes upon addition of one molar equivalent of unlabeled SL2’m are marked with orange (>1 standard deviation) and red (>2 standard deviation) asterisks. All cre26 nucleotides were unambiguously assigned. Conditions: 171 μM cre26, 2 mM MgCl2, 27°C.

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  Figure 7.. Scheme summarizing the new RNA distal interaction mode identified in this study and its possible impact on the HCV life cycle. (A) In a classical kissing loop interaction (top), the bases of two apical loops form Watson–Crick pairs, whereas in the tertiary interaction mode identified in this study, the distal contact involves an apical loop and a base-paired stem. (B) Secondary structure model of the 3’X–5BSL3.2 interaction, together with the possible impact of the different 3’X structures on the HCV life cycle. The 3’X k bases directly involved in the distal contact are base-paired in the upper SL2’ stem, and their complements are exchanged by intermolecular ones upon 5BSL3.2 complex formation. The lower SL2’ stem and subdomain SL1’ remain unaffected, and the rest of the nucleotides forming the upper half of subdomain SL2’ may fold into a hairpin. The switching function of the 3’X domain is probably based on the fact that the DLS is exposed in the isolated domain but blocked upon 5BSL3.2 complex formation. In addition, the unusual structure of the 3’X–5BSL3.2 ‘kissing junction’ proposed in this model may be a platform for protein binding.