Mutations Designed by Ensemble Defect to Misfold Conserved RNA Structures of Influenza A Segments 7 and 8 Affect Splicing and Attenuate Viral Replication in Cell Culture
Ensemble defect designed mutations are indicated by arrows in A and C and by boxes in B and D. Wild-type (WT) sequences are from influenza A/Puerto Rico/1934 H1N1 (PR8) strain. Predicted free energies of folding at 37°C for WT and mutated sequences are provided below each structure. For hairpins and pseudoknots, respectively, ΔG°37 was predicted by RNAstructure  and ShapeKnots programs . (A) The three-way multi-branch loop, 7MB, 79 nts downstream from the 5′ splice site in segment 7 . (B) The predicted folding of 7MB_ED. (C) Pseudoknot/ hairpin equilibrium at the 3′ splice site in segment 7 . (D) The predicted folding of 7PK_ED.