Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand

Abstract

Oligoribonucleotides that corresponded to the X regions of the (+) and (-) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb2+-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such 'structural fingerprinting' conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(-) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.

Figure 1

Secondary structure models of the RNA X(+) and RNA X(−). The sequences correspond to the highly conserved X regions of the (+) and (−) polarity strands of HCV RNA. The RNA X(+) forms a three-stem-loop structure proposed earlier in the literature, the RNA X(−) is arranged into an analogous, hypothetical model. In the figure, the propensity of a given base to be single-stranded (ss-count value) was also shown. The ss-count values were calculated using mfold 3.1 computer program and all folds within 10% of the optimal free energy. The nucleotide symbols have different sizes: the largest symbols indicate that particular nucleotides are single-stranded in all folds (ss-count values of 1), while the smallest symbols show that in all folds nucleotides are paired (ss-count values of 0).

Figure 2

Probing of the structure of RNA X(+) and oligomers: SL3, SL2 and SL2ab by Pb²⁺-induced cleavage and enzymatic digestion methods. (A) Autoradiograms of cleavage of the RNA X(+). The reactions were carried out with [5′-³²P]labeled RNA at 37°C with 0.5, 1 and 2 mM Pb²⁺ ions for 10 min or for 5, 15 and 30 min with ribonuclease T1 and nuclease S1. Lanes: Ci, reaction control; L, formamide ladder; T, limited hydrolysis by RNase T1. Guanine residues are labeled on the right. For probing with Pb²⁺, the short and long run of the gel is shown. (B) Cleavages induced in the RNA X(+) and oligomers: SL3, SL2 and SL2ab by Pb²⁺ ions (left panel), ribonuclease T1 and nuclease S1 (right panel). Cleavages are displayed on the secondary structure models, which are most consistent with experimental data. Relative intensities of Pb²⁺ cleavages are marked as follows: dotted lines, weak; lines, strong; black triangles, very strong cleavages. Enzymatic cleavages are denoted by dotted lines, lines or arrows, corresponding to weak, strong and very strong cleavages, respectively. The symbols are additionally marked with open circles or short, perpendicular lines, for RNase T1 and nuclease S1 cleavages, respectively.

Figure 3

Chemical probing of the SL2 region of RNA X(+) and oligomers SL2 and SL2ab. (A) Probing of adenine residues with DEPC and cytosine residues with DMS; [3′-³²P]end-labeled RNA was used. Lanes: Ci, reaction control; L, formamide ladder; T, limited hydrolysis by RNase T1. In the autoradiogram the modified residues are marked with dots and guanine residues are labeled on the right. (B and C) Correlation of the predicted secondary structures with the experimental data. Relative intensities of strong, weak and very weak modification of adenine residues with DEPC and cytosine residues with DMS are marked by circles with decreasing shadowing. (D) Hypothetical structural rearrangement of the SL2ab region into a pseudoknot structure (see text for details).

Figure 4

Probing of the structure of RNA X(−) and oligomers: SL3(−) and SL2(−) by Pb²⁺-induced cleavage and enzymatic digestion methods. (A) Autoradiograms of cleavage of the RNA X(−). The reactions were carried out with [5′-³²P]labeled RNAs at 37°C with 0.5, 1 and 2 mM Pb²⁺ ions for 10 min or for 5, 15 and 30 min with ribonuclease T1 and nuclease S1. Lanes: Ci, reaction control; L, formamide ladder; T, limited hydrolysis by RNase T1. Guanine residues are labeled on the right. The short and long runs of the gels are shown. (B) Cleavages induced in the RNA X(−) and oligomers: SL3(−) and SL2(−) by Pb²⁺ ions (left panel), ribonuclease T1 and nuclease S1. Cleavages are displayed on the secondary structure models, which are most consistent with experimental data. Relative intensities of Pb²⁺ cleavages are marked as follows: dotted lines, weak; lines, strong; black triangles, very strong cleavages. Enzymatic cleavages are denoted by dotted lines, lines or arrows, corresponding to weak, strong and very strong cleavages, respectively. The symbols are additionally marked with open circles or short, perpendicular lines, for RNase T1 and nuclease S1 cleavages, respectively.