Coronaviruses Resistant to a 3C-Like Protease Inhibitor Are Attenuated for Replication and Pathogenesis, Revealing a Low Genetic Barrier but High Fitness Cost of Resistance
Coronaviruses Resistant to a 3C-Like Protease Inhibitor Are Attenuated for Replication and Pathogenesis, Revealing a Low Genetic Barrier but High Fitness Cost of Resistance
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DOI：
10.1128/jvi.01528-14
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发表时间：
2014-10-01
影响因子：
5.4
通讯作者：
Baker, Susan C.
中科院分区：
文献类型：
--
作者：
Deng, Xufang;Sarah, E. St. John;Baker, Susan C.
病毒蛋白酶抑制剂在阻断人类免疫缺陷病毒和丙型肝炎病毒等病毒的复制方面非常有效，但它们不可避免地导致抑制剂耐药突变体的选择，这可能导致持续的疾病。阻断冠状病毒（CoV）复制的蛋白酶抑制剂，包括严重急性呼吸综合征（SARS）和中东呼吸综合征（MERS）的病原体，为开发抗冠状病毒治疗药物提供了有希望的基础。然而，抗抑制剂冠状病毒的选择和后果尚不清楚。在这项研究中，我们利用模型冠状病毒——小鼠肝炎病毒（MHV），研究了对广谱CoV 3c样蛋白酶（3CLpro）抑制剂产生抗性的MHV准种的基因型和表型。克隆测序鉴定出抗抑制剂病毒3CLpro编码序列的单或双突变。利用反向遗传学方法产生具有突变体3CLpros的等基因病毒，我们发现编码双突变体3CLpros的病毒对该抑制剂具有完全抗性，并且在病毒复制酶多蛋白的蛋白水解过程中表现出明显的延迟。抗抑制剂病毒也表现出延迟和减少传染性病毒颗粒的产生。生化分析证实，双突变体3CLpro酶的蛋白酶活性受损，对抑制剂的敏感性降低，抑制剂水解和活性恢复的动力学延迟。此外，抗抑制剂病毒在小鼠中被证明是高度减毒的。我们的研究首次揭示了抗3CLpro抑制剂的冠状病毒突变体的致病性和机制，揭示了低遗传屏障但高适应成本的抗性。erna病毒因其在选择性压力下（如抗病毒药物的存在）的进化能力而臭名昭著。对于冠状病毒，如中东呼吸综合征（MERS）的病原体，已经开发出蛋白酶抑制剂并显示可以阻止病毒复制，但尚未研究选择具有抑制剂抗性的突变体的后果。在这里，我们报告了冠状病毒模型系统小鼠肝炎病毒（MHV）中冠状病毒对3CLpro蛋白酶抑制剂耐药的低遗传屏障和相对较高的有害后果。我们发现，尽管产生耐药性的突变很快出现，但耐药病毒复制缓慢，不会在小鼠中引起致命疾病。总的来说，我们的研究首次分析了对CoV 3CLpro抑制剂的低屏障但高成本的抗性，这将促进蛋白酶抑制剂作为抗冠状病毒治疗药物的进一步开发。
Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance.IMPORTANCERNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.