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-N 4-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-N 4-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.

中文翻译：

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小分子抗病毒药物 β-dN 4-Hydroxycytidine 抑制具有高抗性遗传屏障的校正完整冠状病毒。

冠状病毒 (CoV) 已从动物宿主中出现，可在人类中引起严重和致命的疾病，但目前尚无 FDA 批准的抗病毒药物来治疗感染。一类抗病毒化合物，核苷类似物，模拟天然存在的核苷以抑制病毒复制。虽然这些化合物已成功治疗多种病毒感染，但诱变核苷类似物，如利巴韦林和 5-氟尿嘧啶，在抑制 CoV 方面无效。这归因于病毒 3'-5' 外切核糖核酸酶 (ExoN) 的校对活性。β-dN 4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) 最近被报道可以抑制多种病毒。在这里，我们证明 NHC 抑制鼠肝炎病毒 (MHV)（50% 有效浓度 [EC50] = 0. 17 μM) 和中东呼吸综合征冠状病毒 (MERS-CoV) (EC50 = 0.56 μM)，具有最小的细胞毒性。NHC 抑制缺乏 ExoN 校对活性的 MHV，类似于野生型 (WT) MHV，表明其具有逃避或克服 ExoN 活性的能力。NHC 仅在感染早期添加时才抑制 MHV，降低病毒特异性感染性，并增加单次感染后存在的 G:A 和 C:U 转换突变的数量和比例。低水平的 NHC 抗性很难实现，并且与 MHV 和 MERS-CoV 基因组中的多个过渡突变有关。这些结果指出了 CoV 中 NHC 抑制的病毒诱变机制，并表明 NHC 抗性的高遗传屏障。一起，数据支持 NHC 用于治疗 CoV 的进一步发展，并提出了 NHC 与 CoV 复制复合物相互作用的新机制，这可能揭示复制的关键方面。它们的流行能力、大流行潜力和引起严重疾病的能力。然而，还没有抗病毒药物被批准用于治疗这些感染。在这里，我们证明了广谱核糖核苷类似物 β-dN 4-羟基胞苷 (NHC) 对两种不同的 CoV 的有效抗病毒活性。病毒校对活动不会显着影响对 NHC 抑制的敏感性，这表明核苷类似物抑制剂和 CoV 复制酶之间存在新的相互作用。更多，NHC 存在下的传代仅产生低水平的抗性，可能是由于多个潜在有害过渡突变的积累。总之，这些数据支持 NHC 抑制的诱变机制，并进一步支持 NHC 用于治疗 CoV 感染的开发。