Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3Cpro), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3Cpro and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation in vivo Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.IMPORTANCE Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.

中文翻译：

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基于活性的蛋白质分析将 ATG4B 确定为肠道病毒 71 增殖的关键宿主因子。


病毒编码的蛋白酶在肠道病毒 71 (EV71) 的有效复制中发挥着多种作用，肠道病毒是人类手足口病 (HFMD) 的病原体。然而，尚不清楚宿主蛋白酶如何影响病毒增殖。在这里，我们设计了基于主要 EV71 蛋白酶 (3Cpro) 抑制剂的活性探针 (ABP)，该蛋白酶负责 EV71 多蛋白的水解，并成功鉴定了与 ABP 结合的宿主候选者。在候选者中，宿主半胱氨酸蛋白酶自噬相关蛋白4同源物B (ATG4B)是自噬机制的关键组成部分，被证明可以水解处理EV71 3Cpro的底物，并且具有与病毒蛋白酶相当的活性。 ATG4B 的基因破坏证实该酶对于体内病毒增殖是不可或缺的。我们的结果不仅进一步加深了对 EV71 生物学中宿主-病毒相互作用的理解，而且还为使用基于活性的蛋白质组学揭示宿主-病原体相互作用提供了样本。重要性 肠道病毒 71 型 (EV71) 是人类手足口病的主要病原体之一，已在世界范围内引起疫情爆发。 EV71 如何通过与多种宿主因子的精心相互作用来有效评估其生命周期仍有待阐明。在这项工作中，我们解构了宿主 ATG4B 蛋白利用其半胱氨酸蛋白酶活性处理病毒多蛋白，并通过化学生物学策略帮助 EV71 复制。我们的结果不仅进一步加深了对 EV71 生命周期的理解，而且还为使用基于活性的蛋白质组学揭示宿主与病原体的相互作用提供了样本。