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

Abstract

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports 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 reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations was now found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless – occasionally – reversion had first occurred. Only a single mutant was viable, likely because its D191E substitution is highly conservative. Remarkably, also a SARS-CoV-2 ExoN knockout mutant was found unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study strongly suggests CoV nsp14 ExoN to have an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that – based on previous MHV and SARS-CoV studies – was proposed to boost longer-term replication fidelity. IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are non-viable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

“…We sought to determine whether nsp14-ExoN activity also contributed to the extensive recombination observed in coronaviruses. Since no proofreading-deficient nsp14-ExoN catalytic mutant is available for MERS-CoV or SARS-CoV-2, we used the MHV nsp14-ExoN inactivation mutant (MHV-ExoN(-)) and wild-type virus (MHV-WT) to compare recombination [ 49 ]. Murine DBT cells were infected with MHV-WT or MHV-ExoN(-) in three independent experiments, and RNA was isolated from infected cell monolayers and viral supernatant when the cell monolayer was intact and 90% cytopathic effect (CPE) was observed.…”

Emergence and Evolution of a Prevalent New SARS-CoV-2 Variant in the United States

“…This mutation occurs at residue 138 in the ExoN domain of nsp14, a novel RNA proofreading domain that ensures the integrity of RNA genomes and transcripts (11). ExoN inactivation is lethal to SARS-CoV-2 (12). The mutation of asparagine (N) to aspartate (D) at this position represents conversion from a neutral to a negatively charged residue in a low complexity domain, possibly involved in mediating protein-protein or RNA-protein interactions(13) ( Figure 3A).…”

“…The mutation of asparagine (N) to aspartate (D) at this position represents conversion from a neutral to a negatively charged residue in a low complexity domain, possibly involved in mediating protein-protein or RNA-protein interactions(13) ( Figure 3A). In addition, nsp14 plays a critical enzymatic role by installing a methyl group on the base of the 5' guanine cap for viral RNA transcripts, which is essential for translation into viral proteins (11,12). Interestingly, the other essential viral factor involved in cap formation on viral transcripts is nsp16, which catalyzes 2'-O-methylation of the nucleotide adjacent to the terminal 7-methyl-guanine cap on viral RNA transcripts (11,14).…”

“…Better resolution of these data through additional sequencing over time should determine the fate and impact of these novel mutations.Biological implications of 20C-US mutationsSeveral mutations carried by the 20C-US variant suggest biochemical or functional impacts on SARS-CoV-2 biology. ORF1b:N1653D is highly unique and specific to the 20C-US lineage.This mutation occurs at residue 138 in the ExoN domain of nonstructural protein (nsp) 14, a novel RNA proofreading domain that ensures the integrity of RNA genomes and transcripts(12).ExoN inactivation is lethal to SARS-CoV-2(13). The mutation of asparagine (N) to aspartate (D) at this position represents conversion from a neutral to a negatively charged residue in a low complexity domain, possibly involved in mediating protein-protein or RNA-protein interactions(14)…”

A Biochemical Perspective of the Nonstructural Proteins (NSPs) and the Spike Protein of SARS CoV-2

“…NSP14 also is reported to have exoribonuclease activity. Inactivating the exoribonuclease (ExoN) activity has been shown to be lethal for SARS CoV-2 [ 39 ].…”