1. Volume 21, Issue 6, Pages 742-753.e8 (June 2017)
Diversity of Functionally Permissive Sequences in the Receptor-Binding Site of Influenza Hemagglutinin 
Nicholas C. Wu, Jia Xie, Tianqing Zheng, Corwin M. Nycholat, Geramie Grande, James C. Paulson, Richard A. Lerner, Ian A. Wilson 
Cell Host & Microbe 
Volume 21, Issue 6, Pages e8 (June 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 742-753. e8DOI: (10. 1016/j. chom. 2017
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3. Figure 1 Profiling of Fitness Effects of Mutations in WSN HA RBS
(A) The locations of mutated residues are indicated on the structure. PDB: 1RVX is used (Gamblin et al., 2004), since it is the only hemagglutinin structure that matches WSN for the amino acid identities at the residues of interest.
(B) The maximum RF index among all mutants that carry the indicated substitution is shown. If all mutants that carry a given substitution are lethal in the deep mutational scanning experiment, the cell in the heatmap that corresponds to that particular substitution is colored in white. If at least one mutant that carries a given substitution is as fit as the wild-type (WT) in the deep mutational scanning experiment, the cell in the heatmap that corresponds to that particular substitution is colored in red. “Single mutants” means the RF index of the indicated single substitution. “Double mutants” means the maximum RF index of all single and double mutants that contained the indicated substitution. “Triple mutants” means the maximum RF index of all single, double, and triple mutants that contained the indicated substitution. The RF index of the amino acid representing the WT sequence is set as 1. For visualization purpose, the maximum RF index is capped at 1.
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4. Figure 2
Experimental Validation and Epistasis in the 220-Loop of WSN HA RBS
(A) The relationship between log10 RF index and log10 TCID50 from virus rescue experiment of WSN mutants is shown as a scatterplot. The green dashed line represents the lower detection limit of the TCID50 assay.
(B) The relationship between log10 RF index and log2 HA titer measured by agglutination of turkey red blood cell is shown.
(C) Representative images of turkey red blood cell agglutination in the presence of WSN virus with increasing dilution factor. With a high dilution factor, red blood cell formed a palette.
(D) The log10 RF index (top) and the log10 TCID50 (bottom) of the indicated double-mutant cycle is shown. A double-mutant cycle includes the wild-type (WT), two single mutants, and the corresponding double mutant. The blue and red lines represent each of the two possible evolutionary pathways from WT to the double mutant (from left to right). The green dashed line represents the lower detection limit of the TCID50 assay.
(E) The left panels represent the double-mutant cycle on wild-type (WT). The right panels represent the double-mutant cycle in the presence of D225E. The TCID50, HA titer, and RF index for each validated WSN variants are listed in Data S1.
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5. Figure 3 WSN Escape Mutants of a Broadly Neutralizing Antibody
(A) The neutralizing activity of C05 IgG against different mutants was measured by a cell viability assay.
(B) The neutralizing activity of S139/1 IgG against different mutants was measured by a cell viability assay.
(C and D) The binding of 50 nM C05 IgG (C) and 50 nM S139/1 IgG (D) to immobilized biotinylated purified HA mutants was measured by Bio-Layer Interferometry (BLI). The raw data (semitransparent) are overlaid with solid lines that represents the best fit model (1:2 bivalent analyte model). The dissociation constants (Kd) are listed in Table S1.
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6. Figure 4 Profiling the Fitness Effects of Mutations in HK68 HA RBS
(A) The maximum RF index of all HK68 mutants that contained the indicated substitution is shown as heatmaps. This is the same analysis as in Figure 1B, but on HK68. The RF index of the amino acid representing the WT sequence is set as 1. For visualization purpose, the maximum RF index is capped at 1.
(B) The relationship between log10 RF index and log10 TCID50 from virus rescue experiment is shown as a scatterplot. The green dashed line represents the lower detection limit of the TCID50 assay.
(C) The relationship between log10 RF index and log2 HA titer measured by agglutination of turkey red blood cell is shown. The TCID50, HA titer, and RF index for each validated HK68 variants are listed in Data S1.
(D) The log10 RF index (top) and the log10 TCID50 (bottom) of the indicated double-mutant cycle is shown. The green dashed line represents the lower detection limit of the TCID50 assay.
(E) The left panels represent the double-mutant cycle on wild-type (WT). The right panels represent the double-mutant cycle in the presence of G225E.
(F) The neutralizing activity of S139/1 IgG against different mutants were measured by a cell viability assay. Data are represented as mean ± SD from three replicates.
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7. Figure 5
Comparison of the Evolutionary Properties of WSN and HK68 RBS 220-Loops
(A and B) The fraction of pairwise substitutions at the indicated residue pairs classified as reciprocal sign epistasis is shown for WSN (A) and HK68 (B). A pair of substitution is classified as interacting with reciprocal sign epistasis when both single substitutions have a lower RF index than WT and the double substitution. Fisher’s exact test was performed to compute the p value.
(C) A total of 3,269 variants were included in the fitness profiling dataset for WSN and HK68. Each variant is denoted by the single-letter codes of amino acids at residues 225, 226, and 228. For each variant, the relationship between the log10 RF index for WSN and for HK68 is shown. Variants carrying a stop codon (nonsense variants) are colored in red. Variants that are included in the validation experiment of both WSN and HK68 are colored in green. Variants that are included in the validation experiment of WSN, but not HK68, are colored in blue. Variants that are included in the validation experiment of HK68, but not WSN, are colored in yellow. Variants corresponding to WT sequence are colored in purple. DQG is the WT sequence for WSN and GLS for HK68.
(D) Virus rescue experiments were performed for nine HK68 HA variants, including WT. However, instead of pairing with HK68 neuraminidase (N2) as in Figure 4, WSN neuraminidase (N1) was used. This experiment was designed to examine the influence of neuraminidase subtype on the fitness of the tested HK68 HA variants.
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8. Figure 6 Structural Characterization of HK68 HA RBS 220-Loop Variants
(A) Comparison of HA RBS backbone conformation between WT (gray) and three variants, namely MTA (lime), LSS (pink), and QAS (cyan). For the WT structure, PDB: 4FNK was used (Ekiert et al., 2012).
(B) Comparison of 220-loop backbone conformation between WT and three variants. Distance between the Cα of residue 225 (sphere representation) in WT and in each variant is indicated.
(C–E) Side chains of residues 225, 226, and 228 and interacting residues (hydrogen bonding) are shown in sticks representation. Hydrogen bonds that are present in the HK68 HA RBS 220-loop variants, but not in the wild-type (WT), are shown in black dashed lines for MTA (C), LSS (D), and QAS (E).
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9. Figure 7
Structural Comparison of HK68 HA RBS 220-Loop Variants with WT
(A) Residue 225 in the crystal structures of wild-type (WT) (Ekiert et al., 2012) and all three variants (MTA, LSS, and QAS) is in a left-handed helix conformation. The side chain of W222 and main chain of residues 224 and 225 are shown in sticks representation. In the left-handed helix conformation, the distance between the backbone amide nitrogen atom of residue 225 to the center of the aromatic ring is around 3.4 to 3.6 Å.
(B) When residue 225 is modeled in right-handed helix conformation, the main-chain carbonyl of residue 224 points toward the plane of the indole ring in W222. These two nucleophilic groups would result in a repulsive force. In this conformation, the distance between the backbone carbonyl oxygen atom of residue 224 to the center of the aromatic ring would be around 2.4 to 2.6 Å.
(C) The Ramachandran plot of residue 225 in the apo form of WT and the three indicated variants are shown. Here, three different crystal structures of WT were analyzed, namely PDB: 4FNK (Ekiert et al., 2012), PDB: 4ZCJ (Lee et al., 2015), and PDB: 4WE4 (Yang et al., 2015). Residue 225 from each HA monomer within the crystal asymmetric unit was analyzed. The orange shading area was created based on data points for the general case of non-Gly (Lovell et al., 2003). Region in white, lighter, and darker orange represents disallowed, allowed, and favored regions, respectively.
(D) The side-chain position and conformation of residue 226 in the crystal structures of WT (PDB: 2YPG) (Lin et al., 2012) and the three indicated variants were compared in the presence of a bound human receptor analog.
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