The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless – in some cases – reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and establishedin vitroassays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV.