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

Abstract

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.

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

“…LAP, LANDO, and LC3/GABARAP-ylation are, to some degree, regulated by the ATG4 family of proteins, and thus the development of ATG4B inhibitors may impact on these processes in addition to the effects on canonical autophagy. Recently, it has been shown that ATG4B proteolytic activity is also essential for replication of enterovirus 71 and can process viral polyprotein, possibly indicating that other cellular targets for this protease exist [21].…”

The regulatory factors and pathological roles of autophagy‐related protein 4 in diverse diseases: Recent research advances

“…Enterovirus 71 protease (EV71) 3C pro is primarily responsible for the hydrolysis of EV71 polyprotein, which is the dominant etiological factor of human hand, foot, and mouth disease (HFMD). Sun et al reported that ATG4B could hydrolyze the substrate of EV71 3C pro and confirmed its indispensable role in viral proliferation through genetic disruption of ATG4B in vivo 142 …”

Dysregulated autophagy contributes to the pathogenesis of enterovirus A71 infection

“…However, several positive-sense, single-stranded RNA viruses (including Dengue virus and hepatitis C virus (HCV)), DNA viruses (such as Varicella-Zoster virus), and even bacteria (Helicobacter pylori or Mycobacterium marinum, for instance) have been demonstrated to activate autophagy to promote their replication/growth cycle at different stages [22][23][24][25]. Following Huang et al 's report that the induction of autophagy in vitro and the formation of autophagosome-like vesicles in vivo after EVA71 infection benefits EVA71 replication [26], increasingly more studies have shown the hijacking of autophagy by EVA71 [10,11,13]. However, one study implied that increased autophagy levels contribute to the inhibition of EVA71 replication [27].…”

“…For early events, the key component of the autophagy machinery autophagy related 4B cysteine peptidase (ATG4B) contributes to the maturation of early viral protein. ATG4B, a cysteine protease that cleaves pro-LC3 isoforms to form LC3-I, may function like EVA71 3C protein to hydrolyze the single polyprotein encoded by the EVA71 genome to generate early viral proteins to undergo the subsequent viral replication process [ 11 , 52 ]. The silencing of ATG4B expression attenuates EVA71 RNA levels and VP1 expression levels in EVA71-infected RD, HEK293T, HeLa, and Vero cells [ 11 ].…”

“…EVA71 is one of the most common etiologic agents that cause hand, foot, and mouth disease (HFMD), which is a highly contagious disease around the world evidence has revealed the hijacking of autophagy and/ or autophagic genes by EVA71 for its replication as well as neurological spread and impairment [10][11][12][13][14]. The corresponding mechanism remains elusive.…”