Fitness barriers limit reversion of a proofreading-deficient coronavirus

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

The rapid evolution of RNA viruses represents a significant challenge for preventing, treating, 57 and eradicating RNA viral diseases. High mutation rates in RNA viruses generate extensive 58 opportunities to overcome evolutionary hurdles, such as antiviral drugs, host immunity, or 59 engineered attenuating changes (1). The evolutionary pathways traversed by RNA viruses are 60 shaped by natural selection, which will favor some evolutionary trajectories more than others 61 based on whether mutations are beneficial, deleterious, or neutral (2). Predicting the likely results 62 of RNA virus evolution is an important step for anticipating viral emergence and for developing 63 escape-resistant antiviral drugs and vaccines (3, 4). 64 65 Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause human illnesses 66 ranging from the common cold to severe and lethal respiratory disease (5). All CoVs encode a 67 proofreading exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is critical for 68 replication, fidelity, fitness, and virulence, and ExoN-inactivation has been proposed as a 69 strategy for live-attenuated vaccine development (6-15). As members of the DEDDh superfamily 70 of exonucleases, CoV ExoNs hydrolyze nucleotides using four metal-coordinating amino acids 71 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 active site mutants in alphacoronaviruses, including transmissible gastroenteritis virus and 77 hCoV-229E, have yet to be recovered and are proposed to be lethal for replication (19, 20) . infections and prolonged passage in tissue culture and following treatment with multiple 87 nucleoside analogues (6-11, 13, 14). SARS-CoV-ExoN-AA also is stable during acute and 88 persistent animal infections in immunocompetent and immune-compromised mice (12) . The lack 89 of primary reversion is not due simply to reduced adaptive capacity, as both SARS-CoV-and 90 MHV-ExoN-AA can adapt for increased replication (7, 14). Most strikingly, long-term passage 91 of MHV-ExoN-AA (250 passages, P250) yielded a highly fit population that had directly 92 compensated for defective proofreading through evolution of a likely high-fidelity RdRp (7 requires mutations to all four sites ( Figure 1A ). Viral mutation rates in the absence of 113 proofreading range from 10 -4 to 10 -6 mutations per nucleotide per round of replication (µ) (1). 114