Small-Molecule Antiviral β-D-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance
Small-Molecule Antiviral β-D-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance
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DOI：
10.1128/jvi.01348-19
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发表时间：
2019-12-01
影响因子：
5.4
通讯作者：
Denison, Mark R.
中科院分区：
文献类型：
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作者：
Agostini, Maria L.;Pruijssers, Andrea J.;Denison, Mark R.
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 批准的抗病毒药物可以治疗这种感染。一类抗病毒化合物，核苷类似物，模仿天然存在的核苷来抑制病毒复制。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' 核糖核酸外切酶 (ExoN) 的校对活性。最近有报道称 beta-D-N-4-Hydroxycytidine (NHC)（EIDD-1931；埃默里药物开发研究所）可抑制多种病毒。在这里，我们证明 NHC 可以抑制小鼠肝炎病毒（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），且细胞毒性最小。 NHC 抑制缺乏 ExoN 校对活性的 MHV，与野生型 (WT) MHV 类似，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能抑制 MHV，降低病毒特异性感染性，并增加单次感染后出现的 G:A 和 C:U 过渡突变的数量和比例。低水平的 NHC 耐药性很难实现，并且与 MHV 和 MERS-CoV 基因组中的多个过渡突变相关。这些结果指出了 CoV 中 NHC 抑制的病毒诱变机制，并表明 NHC 耐药性存在高度遗传障碍。总之，这些数据支持 NHC 治疗 CoV 的进一步发展，并提出了 NHC 与 CoV 复制复合体相互作用的新机制，可能有助于揭示复制的关键方面。 重要性 冠状病毒 (CoV) 从动物宿主进入人类群体，已经证明了它们的流行能力、大流行潜力和引起严重疾病的能力。然而，尚无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-D-N-4-羟基胞苷（NHC）对两种不同的冠状病毒具有有效的抗病毒活性。病毒校对活性不会显着影响对 NHC 抑制的敏感性，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。此外，在 NHC 存在的情况下传代仅产生低水平的抗性，这可能是由于多个潜在有害的过渡突变的积累。总之，这些数据支持 NHC 抑制的诱变机制，并进一步支持 NHC 治疗 CoV 感染的开发。
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). beta-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 mu M) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 mu M) 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, beta-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.