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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基于活动的蛋白质谱分析将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）的有效复制中起着多种作用，肠道病毒71是人类手足口病（HFMD）的病原体。但是，尚不清楚宿主蛋白酶如何影响病毒增殖。在这里，我们基于主要EV71蛋白酶（3Cpro）的抑制剂设计了基于活性的探针（ABP），该抑制剂负责EV71多蛋白的水解，并成功鉴定了与ABP结合的宿主候选物。在候选物中，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，被证明可以水解加工EV71 3Cpro的底物，并具有与病毒蛋白酶相当的活性。ATG4B的遗传破坏证实了该酶对于体内病毒增殖是必不可少的。我们的结果不仅进一步了解了EV71生物学中的宿主-病毒相互作用，而且提供了使用基于活性的蛋白质组学揭示宿主-病原体相互作用的样本。重要事项肠道病毒71（EV71）是人类手足口病的主要病原体之一，已引起全世界的爆发。EV71如何通过与多种宿主因素的精心交互来有效评估其生命周期，尚待阐明。在这项工作中，我们对宿主的ATG4B蛋白具有半胱氨酸蛋白酶活性的病毒多蛋白进行了处理，并通过化学生物学策略帮助EV71复制。