Figure 3.
Targeted disruption of top-ranking IAV mRNA structural motifs potently affects viral fitness. (A) In silico design of a structure-disrupting mutant for IAV segment 4 (HA). Each circle corresponds to a different structure within the Boltzmann ensemble. The diameter of each circle corresponds to the log2 of the relative abundance of the respective structure within the ensemble. Free energies at 37°C and base-pair distances were computed in the absence of any experimental constraint; thus, the predicted structure might slightly differ from the experimentally-determined structure. (B) Targeted DMS-MaPseq analysis of the mutated top-ranking RNA structural motif for segment 4, HA (nt 1607–1670). 90% Winsorizing-normalized reactivities are reported and superimposed on both the WT and on the expected mutant structures. (C) IAV multi-cycle growth kinetics. MDCK cell monolayers were infected with WT or mutant IAVs at a MOI of 10−4. At 14 h post-infection, supernatants were harvested and viral titers determined by plaque assay. P-values are given by Welch's t-test statistics. Error bars correspond to SEs from three biological replicates.