Fitness barriers limit reversion of a proofreading-deficient coronavirus

Kevin W. Graepel; Maria L. Agostini; Xiaotao Lu; Nicole R. Sexton; Mark R. Denison

ExoN-AA reversion, we examined the possible single-nucleotide substitutions surrounding A89 120 and A91 ( Figure 1B ). Three mutations are synonymous, and five mutations yield amino acids 121 unlikely to coordinate with the positively-charged metals required for ExoN catalysis (glycine, 122 valine, proline, threonine, and serine) (16, 19, 21, 22). One mutation per site can restore the 123 acidic charge (i.e. AA-to-ED) but not the native amino acid. These variants have not been tested 124 in a CoV ExoN, but biochemical studies of E. coli DNA polymerase I ExoN mutants suggest that 125 these conservative substitutions would not restore WT-like ExoN activity (23) . We predicted 126 stepwise pathways to ExoN-AAÃ DE reversion based on restoration of acidic charge followed 127 by reversion to native amino acids ( Figure 1C ). We engineered and recovered variants in ExoN-128 AA requiring three mutations (3nt; ExoN-AD, ExoN-EA), two mutations (2nt; ExoN-DA, ExoN-129 ED, ExoN-AE), or one mutation (1nt; ExoN-DD, ExoN-EE) for reversion to WT-ExoN-DE 130 (Table 1) . We will hereafter refer to these mutants as intermediate revertants. All intermediate 131 All rights reserved. No reuse allowed without permission.