Fitness Barriers Limit Reversion of a Proofreading-Deficient Coronavirus

Abstract

The 3=-to-5= exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV-and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants. IMPORTANCE Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.

The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2

“…As reported for MHV and SARS-CoV ExoN mutants ( 23 , 78 ), possible (late) reversion was observed for a few of our MERS-CoV ExoN active-site mutants, specifically, those with E191A, D273E, D273A, and in particular D90E, which had reverted by 6 dpt in four of eight experiments. Together with the immunolabeling results presented in Fig.…”

“…Although the E191D mutant was somewhat more sensitive to the mutagenic agent 5-FU ( Fig. 5C and D ) ( 28 , 78 ), its ExoN activity does not appear to be dramatically altered by this conservative substitution in the active site.…”

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

“…The finding that MHV-ExoN(-) has decreased recombination during viral replication may have important implications for any design of live-attenuated SARS-CoV-2 or other animal or zoonotic CoVs. Our previous studies have shown that the ExoN(-) substitutions in MHV and SARS-CoV are evolutionarily stable over long-term passage in culture and in mice, and that a SARS-CoV ExoN(-) mutant is attenuated in mice while producing a robust and protective immune response against WT SARS-CoV infection [ 38 , 42 , 58 , 59 ]. The results in this paper raise the intriguing possibility that any CoV encoding ExoN(-) would have less recombination potential for repair or escape.…”

“…In another program, rescue of viable ExoN(-) human CoV 229E (HCoV-229E) was unsuccessful, but limited replication was associated with decreased detection of sgmRNAs [34,41]. Although these reports did not study recombination or molecular mechanisms, they support the hypothesis that CoV nsp14-ExoN activity RNA synthesis and possibly recombination, in addition to the known functions of nsp14-ExoN in CoV replication fidelity, viral fitness, in vivo virulence, resistance to nucleoside analogues, and immune antagonism [36,42,43].…”

Imposed mutational meltdown as an antiviral strategy

“…2017, Graepel et al. 2019). Remarkably, however, the compensatory mutations are never back mutations at the altered proofreading positions, nor anywhere else in the proofreading domain.…”