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

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 Pb(2+)-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.