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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/618249 doi: bioRxiv preprint ExoN-DE background. In previous studies, it has been difficult to determine whether the fitness 271 defects in ExoN(-) CoVs are directly linked to low-fidelity replication or through some other 272 mechanism. Our data suggest that the proofreading function of nsp14-ExoN can be uncoupled 273 from its more general role in replication (Figure 4) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/618249 doi: bioRxiv preprint Cloning and recovery of recombinant viruses. Site-directed mutagenesis in MHV genome 306 fragments was performed using "round the horn" PCR (originally described in (33)). Briefly, 307 adjacent primers containing the mutation of interest were 5¢-phosphorylated using T4 308 polynucleotide kinase (NEB, M0201S) using the buffer from the T4 DNA ligase, which contains 309 ATP (M0202S). PCR was performed on a plasmid template using the Q5 High-fidelity 2x previously (9). Briefly, genomic RNA was detected with a 5' 6-carboxyfluorescein (FAM) and 341 3' black hole quencher 1 (BHQ-1) labeled probe targeting nsp2 (Biosearch Technologies, 342 Petaluma, CA), and RNA copy number was calculated by reference to an RNA standard derived 343 from the MHV A fragment. Samples were plated in technical duplicate to minimize well-to-well 344 variation. Titers were determined by plaque assay in DBT-9 cells, and specific infectivity was 345 calculated as PFU per supernatant genomic RNA copy. 346 347 5-fluorouracil sensitivity assays. Stock solutions of 5-fluorouracil (Sigma F6627) were 348 prepared in dimethyl sulfoxide (DMSO). Sensitivity assays were performed in 24-well plates at 349 All rights reserved. No reuse allowed without permission.