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. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex.

Importance: RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

FIG 1

Homology modeling of CoV nsp12-RdRp and identification of residues that potentially regulate fidelity based on CVB3 structure. (A) Phyre2 software was used to model a subsection of the MHV nsp12-RdRp core domain (expanded from the nsp12-RdRp full schematic). (B and C) The modeled MHV RdRp structure (B) was aligned with the solved CVB3 RdRp structure (C). (A and B) The residues chosen for site-directed mutagenesis were selected by comparing previously determined fidelity-altering mutations of picornavirus RdRps. (D) Amino acid alignments across CoVs showing that all residues are almost completely conserved.

FIG 2

Resistance of MHV nsp12-RdRp mutant viruses to 5-fluorouracil in the WT and nsp14-ExoN(−) backgrounds. The domain locations of mutations are indicated as follows: fingers, blues; palm, reds. DBT cells were pretreated with different concentrations of 5-FU for 30 min. The treatment was removed, and the cells were infected with the indicated viruses in the WT background (A) or the nsp14-ExoN(−) background (B) at an MOI of 0.01. The medium containing 5-FU was replaced 30 min p.i. Virus samples were taken at 24 (WT) or 32 [nsp14-ExoN(−)] h p.i., and the titer was determined by plaque assay. The data represent the results of 3 independent experiments, each with 2 replicates. The error bars represent standard errors of the mean (SEM) *, P < 0.05 by the Wilcoxon test.

FIG 3

Replication kinetics of MHV nsp12-RdRp mutant viruses. The mutation location is indicated as follows: fingers, blues; palm, red. DBT cells were infected with the viruses indicated in the WT background (A, C, and E) or the nsp14-ExoN(−) background (B, D, and F) at an MOI of 0.01 PFU/cell. Supernatant aliquots were taken at the indicated times p.i., and titers were determined by plaque assay. Total RNA was taken at the indicated times p.i., and RT-qPCR was performed. The data represent the results of 3 independent experiments. The error bars represent SEM.

FIG 4

Specific infectivity is increased in both nsp12-V553I and nsp12-M611F mutants. DBT cells were pretreated with increasing concentrations of 5-FU for 30 min. The treatment was removed, and the cells were infected with the indicated viruses in the WT background (A) or the nsp14-ExoN(−) background (B) at an MOI of 0.01. The medium containing 5-FU was replaced 60 min p.i. Virus samples were taken at 20 and 24 h p.i. Titers were determined by plaque assay, and the numbers of supernatant genomes were determined using one-step RT-qPCR. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM (*, P < 0.05 by 2-way analysis of variance [ANOVA] using the Bonferroni correction for multiple comparisons).

FIG 5

Competitive fitness analysis in the nsp14-ExoN(−) background. DBT cells were pretreated with medium alone or medium containing 60 μM 5-FU for 30 min. The treatment was removed, and the cells were coinfected at a total MOI of 0.01 with nsp14-ExoN(−) and nsp12-V553I/nsp14-ExoN(−) (A) or nsp12-M611F/nsp14-ExoN(−) (B) viruses at a ratio of 9:1, 1:1, or 1:9. Medium alone or containing 60 μM 5-FU was replaced 30 min p.i. Total RNA was taken at 24 h p.i. Sequencing was performed across a 1.7-kb region of nsp12-RdRp that included both mutations. The data represent the results of 3 independent experiments, each with 2 replicates. The error bars represent SEM.

FIG 6

The nsp12-V553I mutation is stable across passages; however, nsp12-M611F is vulnerable to reversion. DBT cells were infected at an initial MOI of 0.01 and then blind passaged in triplicate for 5 passages. Total RNA was taken, and sequencing was performed across a 1.7-kb region of nsp12-RdRp that included both mutations. The percentage of each nucleotide present in each of the triplicate lineages after 5 passages is shown. Mutant viruses in the WT and nsp14-ExoN(−) backgrounds are shown. The original mutation for each of the viruses is shown above the graph, and the likely majority, secondary and tertiary codons present in the population are shown below the graph.

FIG 7

Resistance of MHV nsp12-RdRp V553I and M611F mutant viruses to 5-azacytidine in the nsp14-ExoN(−) background. The domain locations of mutations are indicated as follows: fingers, blues; palm, reds. DBT cells were pretreated with different concentrations of 5-AZC for 30 min. The treatment was removed, and the cells were infected with the indicated viruses at an MOI of 0.01. The medium containing 5-AZC was replaced 30 min p.i. Virus samples were taken at 32 h p.i., and the titer was determined by plaque assay. The data represent the results of 5 independent experiments, each with 2 replicates. The error bars represent SEM (*, P < 0.05 by ratio paired t test).

FIG 8

The nsp12-V553I mutation confers decreased accumulation of mutations in the nsp14-ExoN(−) background with no bias toward the exclusion of specific nucleotides. DBT cells were infected at an MOI of 0.01, and total RNA was collected. Deep sequencing was performed on the samples. The statistically significant mutations present at ≥1% of the total population are shown for the wild type, nsp14-ExoN, and nsp12-V553I or nsp12-M611F in both backgrounds. They are graphed according to their distribution across the genome (A to F), with intentionally introduced mutations shown with circles colored blue (nsp12-V553I), red (nsp12-M611F), or green [nsp14-ExoN(−)] (B to F); as the total number of mutations present in the population (G); and as the percentage of specific mutations present (H).