Small-Molecule Antiviral β-d- N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance

Abstract

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). β-d-N⁴-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC₅₀] = 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC₅₀ = 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N⁴-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

Keywords: MERS-CoV; RNA-dependent RNA polymerase; RdRp; SARS-CoV; antiviral resistance; coronavirus; nucleoside analogue; pandemic.

FIG 1

Chemical structure of EIDD-1931, β-d-N⁴-hydroxycytidine.

FIG 2

NHC inhibits MHV and MERS-CoV with minimal cytotoxicity. (A and B) Changes in MHV (A) and MERS-CoV (B) titers relative to vehicle control after treatment with increasing concentrations of NHC. The data represent the results of 6 independent experiments, each with 3 replicates. The error bars represent standard errors of the mean (SEM). (C) Changes in titer data from panel A, represented as percentages of that of vehicle control. WT MHV, EC₅₀ = 0.17 μM. (D) Changes in titer data from panel B, represented as percentages of that of vehicle control. WT MERS-CoV, EC₅₀ = 0.56 μM. (E) DBT-9 cell viability as a percentage of that of DMSO control across NHC concentrations. No cytotoxicity was detected up to 200 μM. The data represent the results of 2 independent experiments, each with 2 replicates (MHV). The error bars represent SEM. (F) Vero cell viability as a percentage of that of DMSO control across NHC concentrations. Less than 50% cytotoxicity was detected up to 10 μM. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM.

FIG 3

The NHC inhibition profile of MHV is consistent with mutagenesis. (A) Treatment with 16 μM NHC (∼100 times the EC₅₀) significantly inhibits MHV replication during a single infection when added before 6 h p.i. (B) Both MHV titer and monolayer RNA copies decrease after treatment with increasing concentrations of NHC. (C) NHC treatment results in a decrease in supernatant MHV RNA. (D) Data from panel C represented as the ratio of infectious WT MHV to genomic MHV RNA present in the supernatant, or specific infectivity, normalized to that of vehicle control. NHC treatment resulted in a decrease in the specific infectivity of MHV. All the data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM. Statistical significance compared to DMSO control was determined by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test for multiple comparisons. *, P < 0.05; **; P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

NHC treatment drives an increase in low-frequency G:A and C:U transition mutations in WT MHV during a single infection. (A to C) Distribution and frequencies of variants across the genome detected by NGS after vehicle treatment (A), 2 μM NHC treatment (B), or 4 μM NHC treatment (C). The log₁₀ depth of coverage at each genomic position is depicted by the lines; the frequencies of individual mutations spread across the genome are represented by the dots. (D to F) Numbers of mutations in WT MHV after infection in the presence of vehicle (D), 2 μM NHC (E), or 4 μM NHC (F) presented by type. Transition mutations are shown in gray, and transversion mutations are shown in white. (G and H) Changes in relative proportions of each mutation type after treatment with 2 μM NHC (G) or 4 μM NHC (H) compared to vehicle control. The relative proportions of G:A and C:U transitions increased with increasing concentrations of NHC treatment and are indicated by green shading.

FIG 5

Sensitivity of ExoN(−) MHV to inhibition by NHC. (A) Changes in viral titers for WT MHV and ExoN(−) MHV relative to vehicle control after treatment with NHC. ExoN(−) MHV is more sensitive to NHC than WT MHV. The data represent the results of 3 independent experiments, each with 3 replicates. The error bars represent SEM. Statistical significance compared to WT MHV was determined by a Wilcoxon test. **, P < 0.01. (B) Changes in viral titer data from panel A represented as a percentage of that in vehicle control. WT, EC₉₀ = 1.59 μM; ExoN(−), EC₉₀ = 0.72 μM. ExoN(−) MHV is approximately 2-fold more sensitive to NHC than WT MHV.

FIG 6

Resistance and mutational profiles of MHV after 30 passages in the presence of NHC. (A) Changes in viral titers for WT MHV, MHV p30.1, and MHV p30.2 relative to vehicle controls after treatment with NHC. MHV NHC p30.2 was less sensitive to NHC than WT MHV, while MHV p30.1 showed no change in sensitivity. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM. Statistical significance compared to WT MHV was determined by ratio paired t test. *, P < 0.05. (B) Changes in viral titer data from panel A represented as percentages of that of vehicle control. WT MHV, EC₉₀ = 1.53 μM; MHV p30.1, EC₉₀ = 2.61 μM; MHV p30.2, EC₉₀ = 2.41 μM. (C) Replication kinetics of NHC passage viruses. MHV p30.1 and p30.2 were delayed in replication compared to WT MHV but ultimately reached similar peak titers. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent standard deviations (SD). (D) MHV p30.1 accumulated a total of 162 consensus mutations across the genome that were detectable by Sanger sequencing. Of these mutations, 81 were synonymous, 64 were nonsynonymous, and 17 were noncoding. (E) MHV p30.2 accumulated 102 total mutations across the genome. Of these mutations, 54 were synonymous, 42 were nonsynonymous, and 7 were noncoding. (F) Each lineage accumulated more synonymous changes than nonsynonymous or noncoding changes over passage. (G) Breakdown of transition and transversion mutations present in each lineage after passage. MHV p30.1 and p30.2 mutations were predominantly transitions. (H) Breakdown of the types of transition mutations present in each lineage across passage. G:A transitions were the most abundant for both MHV p30.1 and p30.2.

FIG 7

Resistance and mutational profiles of MERS-CoV after 30 passages in the presence of NHC. (A) Changes in viral titers relative to vehicle controls after treatment with NHC for WT MERS-CoV passaged 30 times in the absence of drug, MERS-CoV p30.1, and MERS-CoV p30.2 relative to vehicle controls after treatment with NHC. Both MERS-CoV p30.1 and p30.2 were less sensitive to NHC than WT MERS-CoV. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM. (B) Changes in viral titer data from panel A represented as percentages of that of vehicle control. WT MERS-CoV, EC₉₀ = 1.31 μM; MERS-CoV p30.1, EC₉₀ = 3.04 μM; MERS-CoV p30.2, EC₉₀ = 2.12 μM. (C) Replication kinetics of NHC passage viruses. WT MERS-CoV, MERS-CoV p30.1, and MERS-CoV p30.2 replicated with similar kinetics and reached similar peak titers. The data represent the results of 2 independent experiments, each with 3 replicates. The error bars represent SEM. (D) MERS-CoV p30.1 accumulated 27 total mutations across the genome. Of these mutations, 14 were synonymous and 13 were nonsynonymous. (E) MERS-CoV p30.2 accumulated 41 total mutations. Of these mutations, 17 were synonymous and 24 were nonsynonymous. (F) Both MERS-CoV p30.1 and p30.2 accumulated similar numbers of nonsynonymous and synonymous changes during passage. (G) MERS-CoV p30.1 and p30.2 acquired predominantly transitions. (H) Types of transition mutations present in each lineage across passage. MERS-CoV p30.1 acquired more G:A transitions, whereas MERS-CoV p30.2 acquired similar numbers of each transition type.