Fitness barriers limit reversion of a proofreading-deficient coronavirus

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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/618249 doi: bioRxiv preprint Our results also extend existing studies of CoV ExoN motif I. Motif I AAÃ DE mutations in the 249 SARS-CoV nsp14-ExoN dramatically reduce nuclease activity in biochemical assays, but no 250 study has examined the contributions of each residue independently (16, 18) . Intermediate 251 revertants of ExoN-AA did not display consistent or statistical differences in replication, 5-FU 252 sensitivity, or competitive fitness relative to ExoN-AA, suggesting that they remain 253 phenotypically ExoN(-) during infection and supporting previous studies that motif I DE is 254 essential for WT ExoN function. Given these results, we were surprised to observe repeated 255 reversion of the ExoN-AE but not the other two 2nt variants, ExoN-DA and ExoN-ED. One 256 potential explanation is that the specific mutational bias of ExoN-AE makes the revertant 257 mutations more accessible than in ExoN-DA or ExoN-ED. Alternatively, if ExoN-AE has 258 profound replication or fitness defects, selection could drive primary reversion more quickly 259 away from this genotype. Consistent with this hypothesis, ExoN-AE reverted more quickly at a 260 higher MOI, where natural selection acts more efficiently on a larger population size (Table 1) Consistent with this hypothesis, the fitness effects of mutations in nsp12-P250 and nsp14-P250 269 differ based on the motif I genotype; they are beneficial in ExoN-AA but detrimental in the WT-270 All rights reserved. No reuse allowed without permission.