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.

Nidovirus ribonucleases: Structures and functions in viral replication

“…For SARS-CoV and Murine hepatitis virus (MHV)-A59, by contrast, viable ExoN active-site mutants could be obtained, even though they also displayed defects in RNA synthesis and virus reproduction. 67,68 Consistent with a role of ExoN in proofreading, replication fidelity of MHV-A59 and SARS-CoV encoding substitutions of residues critical for ExoN activity was found to be reduced about 15 and 21-fold compared to wildtype MHV and SARS-CoV, respectively. More detailed analysis of SARS-CoV revealed that the defect led to higher diversity ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

“…No statistically significant increase in diversity between passages could be observed, suggesting selection of advantageous (and counterselection of deleterious) mutations during virus propagation in cell culture. Eckerle et al 67 calculated a higher-than-average replication fidelity of SARS-CoV and MHV (9.0 x 10 -7 and 2.5 x 10 -6 substitutions, respectively, per nucleotide per replication cycle) when compared to other RNA viruses, while the average replication fidelity of SARS-CoV and MHV mutants encoding substitutions of ExoN active-site residues was similar to that calculated for other RNA viruses. Although these calculations probably underestimate mutation frequencies as the studies were performed with infectious viruses (counterselecting mutants with non-viable genotypes), they provide convincing evidence for ExoN playing a major role in securing a relatively high replication fidelity, similar to that reported for small DNA viruses.…”

“…69 More accurate estimates of replication fidelities may be derived from future studies in which coronavirus replicases are expressed from RNAs that are distinct from the substrate RNAs used to measure mutation frequency. Taken together, the (reverse) genetic studies by Eckerle et al 67,68 provide circumstantial evidence to support a function of ExoN as a proofreading exoribonuclease. Biochemical evidence to support this hypothesis remains to be obtained, for example by comparing the fidelity of RNA synthesis by purified RdRp (nsp12 and co-factors) in the presence and absence of ExoN.…”

Ultradeep Sequencing Analysis of Population Dynamics of Virus Escape Mutants in RNAi-Mediated Resistant Plants

“…On the other hand, next-generation sequencing (NGS) techniques can generate massive amounts of genetic data, which can be used to detect variants at much lower frequencies (Wang et al 2007;Eriksson et al 2008;Solmone et al 2009;Cordey et al 2010;Eckerle et al 2010;Murcia et al 2010;Zagordi et al 2010;Willerth et al 2010;Bunnik et al 2011;Guo et al 2011;Wrigth et al 2011). In this study, we have generated tens of millions of short reads with Illumina sequencing to obtain an unprecedented ultradeep coverage of amiR targets in the TuMV genome, thus characterizing in great detail the genetic composition of TuMV lineages evolving in WT and 10-4 plants at different time points during the experimental evolution process and right after the first successful infection of 12-4-resistant plants.…”

“…Although in general the limited length of Illumina reads makes it difficult to assess linkage among mutations, in our particular case this was not an issue because the 21-nt sequences corresponding to the amiR159-HC-Pro target were completely covered by the 76-nt-long reads. The validity of the Illumina technology for assessing virus diversity has been proved with studies of the severe acute respiratory syndrome coronavirus (Eckerle et al 2010), human rhinovirus (Cordey et al 2010), HIV-1 (Willerth et al 2010), and foot-and-mouth disease virus (Wrigth et al 2011). …”