Figure 1. Summary of experimental conditions and data normalization

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Figure 1. Summary of experimental conditions and data normalization Figure 1. Summary of experimental conditions and data normalization. (A) Schematic of the experimental workflow. (B) ... Figure 1. Summary of experimental conditions and data normalization. (A) Schematic of the experimental workflow. (B) Sample DMS-MaPseq reactivities from a hairpin-forming region of IAV segment 8, NS (nt 409–435). The two halves of the helix (indicated by dashed lines) have lower DMS reactivities compared to the loop region. (C) Heatmap of pairwise Pearson correlations for all IAV DMS-MaPseq datasets. Significant correlations (P-value < 0.05) are denoted by a star in the upper-right corner. (D) Violin plot of normalized (90% Winsorizing) DMS reactivities across IAV transcriptome. The median of each distribution is indicated. (E) (left) Snapshot of 90% Winsorizing and 2-step normalized DMS reactivities on the first 550 nt of E. coli 16S rRNA under both denaturing and native deproteinized ex vivo conditions. (right) 2-step normalized DMS reactivities superimposed onto the phylogenetic-derived secondary structure of the E. coli 16S rRNA. The inlay shows superimposition of 90% Winsorizing-normalized reactivity values of the boxed domain for comparison. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz318, https://doi.org/10.1093/nar/gkz318 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Transcriptome-wide in vivo secondary structure model of IAV mRNAs. In vivo and in vitro DMS reactivities, ... Figure 2. Transcriptome-wide in vivo secondary structure model of IAV mRNAs. In vivo and in vitro DMS reactivities, reactivity difference (ΔDMS), Shannon entropies, base-pairing probabilities, minimum expected accuracy (MEA) structure, and helix models with superimposed in vivo DMS reactivities for IAV segment 8, NS (NS1/NEP). Reactivity values are reported as the arithmetic mean of two biological replicates. Error bars represent SDs. Base-pairs are depicted as arcs, colored according to their probabilities. Green arcs correspond to base-pairs with P ≥ 0.7. Black dashed arcs correspond to pseudoknots. Regions with multiple overlapping arcs (high Shannon entropies) correspond to regions that are likely to form alternative structures. Base-pairs in the MEA structure are depicted as arcs, colored according to their conservation as determined by multi-sequence alignment of IAV mRNAs. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz318, https://doi.org/10.1093/nar/gkz318 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Targeted disruption of top-ranking IAV mRNA structural motifs potently affects viral fitness. (A) In silico ... 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 log<sub>2</sub> 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<sup>−4</sup>. 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz318, https://doi.org/10.1093/nar/gkz318 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Spontaneous reversion of segment 4 mutant IAV Figure 4. Spontaneous reversion of segment 4 mutant IAV. (A) Schematic of the in vitro IAV evolution experiment. (B) ... Figure 4. Spontaneous reversion of segment 4 mutant IAV. (A) Schematic of the in vitro IAV evolution experiment. (B) Selected structural motif for segment 4. Bases that were altered by targeted mutagenesis are indicated in red. Spontaneous mutations appearing during the time course of in vitro evolution are indicated in green. (C) IAV multi-cycle growth kinetics. MDCK cell monolayers were infected with WT or mutant 4 IAVs (either from passage 0 or 12 of the in vitro evolution experiment, replicate #1) at a MOI of 10<sup>−4</sup>. 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 3 biological replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz318, https://doi.org/10.1093/nar/gkz318 The content of this slide may be subject to copyright: please see the slide notes for details.