Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

Abstract

Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. RNA virus replication results in the incorporation of a relatively high number of mutations, ranging from 10 Ϫ4 to 10 Ϫ6 mutations per site per round of replication (1-5). It is thought that low-fidelity replication is largely responsible for the capacity of RNA viruses to evolve rapidly and adapt to new host species and ever-changing environmental pressures (6-8). RNA-dependent RNA polymerase (RdRp) is central to the replication of RNA viruses and is a key regulator of nucleotide selectivity and fidelity (9, 10). Recent studies of coxsackievirus virus B3 (CVB3), poliovirus, HIV-1, and other viruses have demonstrated that viable viruses are recoverable only within a 4-fold range of RdRp fidelity (11)(12)(13)(14). In most cases, altered RdRp fidelity decreases fitness relative to wild-type (WT) viruses; this has been demonstrated for changes as small as a 1.2-fold difference in the accumulation of mutations (12,(14)(15)(16). Despite having as little as no amino acid identity outside conserved motifs (11)(12)(13)(14)(17)(18)(19), all described polymerase structures (including RdRps) resemble a "cupped right hand," with finger, palm, and thumb domains (20). The fingers form a channel that allows entry of the template RNA and ribonucleotide triphosphates (rNTPs) and assist in proper positioning of incoming nucleotides in the active site (21). The palm contains the active site, and the thumb functions in contacting exiting nascent RNA (21-23). However, there is diversity in the viral genes that encode RdRps; additional domains that perform a variety of functions, such as methyltransferase, endonuclease, polyribonucleotidyl transferase, guanylyltransferase, membrane targeting, protein-

Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes

“…However, such mutations were more frequently found in the RNA produced under similar conditions by an ExoN knockout mutant (Smith et al, 2013). The observation that even the wt virus incorporates 5-FU is consistent with the finding that nsp12 RdRp domain mutations (see above) can improve the selectivity of the viral RNA polymerase for 5-FU incorporation (Sexton et al, 2016).…”

“…As described for other RdRp domains, the key lysine of motif D, which likely acts as general acid during catalysis (Castro et al, 2009), is located near the entrance of the NTP channel. Further functional predictions about the CoV RNA polymerase come from an alignment of a Phyre2-based molecular model of MHV nsp12 with the crystal structures of the RdRp domains of coxsackievirus B3 (CVB3) and poliovirus (Sexton et al, 2016). In particular, this study identified V553 and M611 (homologs of SARS-CoV nsp12 residues V557 and M615, Fig.…”

“…However, using sequence alignments, secondary structure predictions and homology modelling, the molecular structure of the nsp12 subunit of SARS-CoV (Xu et al, 2003) (Fig. 3B) and MHV (Sexton et al, 2016) has been predicted. In case of the former, a model could be reliably generated for its conserved RdRp domain, based on a careful alignment of amino acids 388-890 to known polymerase sequences (Xu et al, 2003); Fig.…”

“…3F) as putative equivalents of the CVB3 RdRp residues I176 and I230, which are known to be involved in RdRp fidelity. Subsequent mutation of these residues to isoleucine (V553I) and phenylalanine (M611F) in the virus conferred resistance to 5-fluorouracil (5-FU) and 5-azacytidine (5-AZC), or 5-FU only, respectively (Sexton et al, 2016).…”

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

“…Protein subunits of the larger RNAsynthesizing complex, like nsp7-nsp8, the nsp13-helicase, and the nsp14-ExoN, likely exert a strong influence on RdRp behavior and performance. On the other hand, a recent study employing homology modeling and reverse genetics of the MHV RdRp domain described the first two nsp12 mutations that can induce resistance to a mutagen and reduce the MHV RdRp error rate during virus passaging (Sexton et al, 2016). So, not unexpectedly, also features within nsp12 itself contribute to properties like nucleotide selectivity and fidelity regulation.…”