Activity-Based Protein Profiling Identifies ATG4B as a Key Host Factor for Enterovirus 71 Proliferation

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 hostpathogen interactions.