Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing

Abstract

Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution. Quasispecies diversity is critical to virus fitness, adaptation, and pathogenesis. However, the relationship of fidelity to population diversity is less studied because viral systems with engineered differences in fidelity and bioinformatic methods that robustly measure and compare fidelity and diversity during replication and passage have not been available. Coronaviruses contain the largest and most complex RNA genomes, and encode multiple novel replicase nonstructural proteins (nsps). We previously demonstrated that murine hepatitis virus nsp14-exonuclease (ExoN) activity is required for replication fidelity. In the present report we have generated nsp14-ExoN inactivation mutants of SARS-coronavirus (S-ExoN) that have stable growth defects and dramatically decreased replication fidelity during replication in culture. We used the S-ExoN mutant viruses to define the diversity and stability of the genome during replication and passage, and to test the capacity of deep sequencing to track virus population diversity over time. The experiments demonstrate that viable S-ExoN mutants accumulate large numbers of predominantly unique mutations across the genome, and that increased diversity is continuous over passage. The results establish methods for direct comparison of consensus genome sequences with total population diversity and the impact on viral growth and adaptation.