Mutational disruption of the 5BSL3.2 RNA structure blocked HCV RNA replication, revealing an essential kissing-loop interaction between the 3' end of the NS5B coding sequence and the 3' nontranslated region.
Identification of a pseudoknot structure at the 3' end of the HCV genome involving a kissing-loop interaction that is essential for viral RNA replication.
The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for replication.
Friebe et al. (Thu,) conducted a other in Hepatitis C Virus (HCV) infection (in vitro model). Mutational disruption of 5BSL3.2 structure vs. Wild-type HCV replicon was evaluated on HCV RNA replication efficiency (luciferase activity ratio 48h/4h). Mutational disruption of the 5BSL3.2 RNA structure blocked HCV RNA replication, revealing an essential kissing-loop interaction between the 3' end of the NS5B coding sequence and the 3' nontranslated region.