Viral gain-of-function experiments uncover residues under diversifying selection in nature

Rohan Maddamsetti; Daniel T. Johnson; Stephanie J. Spielman; Katherine L. Petrie; Debora S. Marks; Justin R. Meyer

Recall that 97% of substitutions in OmpF + gain-of-function experiments occur in the specificity region. Likewise, the natural J homologs had disproportionate variation here: 30% of the total amino acid variation occurred in the specificity region, despite only being 15% of the total length of J ( Figure 2A ). This nonrandom clustering of variation in the specificity region strongly suggests that J has experienced substantial diversifying selection on host attachment. Furthermore, the 17 residues engineered into the MAGE libraries were significantly more variable than randomly chosen groups of 17 residues from J (non-parametric bootstrap: p < 10 -5 ) and from the specificity region 960-1132 (non-parametric bootstrap: p = 0.0054) showing the experiments had identified evolutionary hotspots. However, the specific 17 amino acid substitutions evolved in the laboratory were not common in natural homologs suggesting that the selection experiments had the resolution to predict where changes would evolve, but not the exact change (Supplementary Figure 1) .