EVIDENCE FOR THE EXISTENCE OF A PSEUDOKNOT STRUCTURE AT THE 3'-TERMINUS OF THE FLAVIVIRUS GENOMIC RNA

The 3'-terminal nucleotides of the flavivirus genomic RNA form conserv ed secondary structures that may function as cis-acting signals for RN A replication. Here we provide evidence for the existence of a conserv ed pseudoknot structure at the 3' terminus of the flavivirus genomic R NA. A truncated version of the West Nile virus (WNV) 3'-terminal RNA s equence was used as the model for these studies, Circular dichroism sp ectra indicated the presence of a highly structured RNA conformation w ith a significant amount of A-form helix. Ribonuclease probing not onl y confirmed the presence of the predicted secondary structure, which c onsists of a long stem-loop (SL1) and a shorter stem-loop (SL2), but a lso suggested that base pairing occurs between nucleotides in the loop of SL2 and those in an internal loop strand located on the 5' side of SL1. Analysis of three mutant RNAs further supported the existence of pseudoknot interactions. UV-melting analysis of the WNV 3' model RNA showed three transitions with significant hyperchromicity at approxima tely 46, 62, and 79 degrees C. UV-melting analysis with either SL1 or SL2 RNA alone suggested that the 62 and 79 degrees C transitions repre sent the unfolding of SL2 and SL1, respectively. The 46 degrees C tran sition is most likely due to the opening of the proposed tertiary stru cture. A similar melting curve was obtained for another flavivirus (de ngue-3 virus) 3'-terminal RNA, providing further support for the conse rvation of the structure among flaviviruses. Molecular modeling of the RNA indicated that a pseudoknot structure is a stereochemically and e nergetically reasonable model for the 3' terminus of flavivirus genomi c RNA.