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.

A Pseudoknot in a Preactive Form of a Viral RNA Is Part of a Structural Switch Activating Minus-Strand Synthesis

“…Transcripts were phenol-chloroform extracted, ammonium acetate (NH 4 OAc)-isopropanol precipitated, and then purified through a 6% polyacrylamide gel electrophoresis (PAGE)-8 M urea sequencing-length gel. Full-length RNA was excised from the gel and the RNA removed from the gel slice by shaking overnight in an equal volume of 0.6 M NH 4 OAc, pH 5.3, 0.1% sodium dodecyl sulfate, and phenolchloroform (1:1, vol/vol), followed by phenol-chloroform extraction and NH 4 OAc-ethanol precipitation. RNA was resuspended in H 2 O. RNA transcripts were quantified with a UV spectrophotometer.…”

“…Changes in viral RNA conformation in 3Ј regions of the genome that hide or expose the 3Ј terminus (13,18,24) or permit the formation of important cis-acting structures (11) likely regulate initiation of minusstrand synthesis. Evidence for important alternative structures with no known function has also been reported (4,5). Since RNA switches usually involve long-distance tertiary interactions that stabilize one of the RNA conformations (8,11,12), removal of cis-acting elements from their natural context for biochemical and biophysical analyses may lead to an oversimplification of how diverse elements are involved in many viral processes.…”

Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate

“…While chemical probing data and mutational analysis both support the existence of the 39SL1 stem-loop at the extreme 39-end (Blight and Rice 1997;Yi and Lemon 2003a;Dutkiewicz and Ciesiolka 2005), the structure adopted by the X55 RNA region (which is 100% conserved in its sequence among different HCV strains) remains controversial. No homologous sequences have been found in related viruses, in phylogenetically distinct viruses, or in cellular mRNAs (Dutkiewicz and Ciesiolka 2005). While the reasons underlying the extraordinarily high level of sequence conservation within the unique X55 region is unknown, it is likely due, at least in part, to the need to maintain higher ordered RNA structures required for essential processes in the viral life cycle.…”

RNA Partial Degradation Problem: Motivation, Complexity, Algorithm

“…RNA-A 108 and RNA-B 66 molecules were obtained and labeled as described in the literature (Dutkiewicz et al, 2005). Briefly, both RNAs were synthesized by in vitro transcription involving chemically synthesized DNA as templates.…”