Figure 6: Functional maps of the C-terminal half of the AAV9 capsid amino acids. | Nature Communications

Figure 6: Functional maps of the C-terminal half of the AAV9 capsid amino acids.

From: Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

Figure 6

(a) A 2D heat map showing correlations between positions of AA mutations and phenotypic changes. Degrees of phenotypic changes caused by AA mutations are categorized into seven groups as indicated to the right. Functionally important amino acids form four clusters (Clusters I–IV) that are partially discontinuous by the presence of the amino acids responsible for the structural integrity shown as grey regions. The following is the orders of the samples in the heatmap (from the top to the bottom): liver (Lv), skeletal muscle, dorsal skin, intestine, brain (B), visceral fat, lung, pancreas, testis, heart (H), kidney and spleen (S) for the tissue samples; Pro5 transduction, Lec2 transduction, Pro5 binding and Lec2 binding for the in vitro samples and 1 min, 10 min, 30 min, 1 h, 4 h, 8 h, 24 h and 72 h time points for the blood samples. No significant phenotypic changes (ns, indicated with yellow) are those showing a greater than fourfold increase or decrease in a PD value with no statistical significance (two-tailed Mann–Whitney U-test, P≥0.05). (b) A 3D map on the full AAV9 capsid atomic model showing topological locations of the functionally important amino acids in Clusters I–IV. A triangle and three pentagons indicate three- and fivefold symmetry axes, respectively. Twofold symmetry axes are in the centre of two adjacent fivefold symmetry axes. The image was generated by PyMOL. (c) A Venn diagram showing correlation between AAV9 capsid amino-acid residues and various AAV9 phenotypes. The AAV9 neutralizing antibody epitope, the amino acids responsible for enhanced liver transduction, and 3 of the 16 amino acids important for postattachment processing, F501, A502 and N515, were identified through the AAV2R585E mutants. All other amino acids and their phenotypes were identified by the AA mutagenesis study of the AAV9 capsid. Owing to the nature of the AA mutagenesis approach, it remains unknown which one of the two amino acids mutated to alanine is responsible for the phenotypes or whether both of the two amino acids are responsible for the phenotypes.

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