Enterovirus 71 (EV71) is the causative agent of hand, foot, and mouth disease (HFMD), which typically affects infants and children (1). HFMD usually presents as a mild febrile disease with a localized rash, but some patients may develop infection of the central nervous system (CNS) with illness ranging from aseptic meningitis through fatal encephalitis (2). In the last decade, outbreaks of HFMD have regularly reoccurred through Asia (3, 4). According to data from the Chinese Center for Disease Control and Prevention (CDC), more than 420,000 cases of HFMD, with 70 deaths, were reported in China in April 2014. Currently, there is no antiviral therapy available for treatment of HFMD.EV71 belongs to the genus Enterovirus in the family Picornaviridae (5-7). Similar to other picornaviruses, EV71 contains a single-stranded, positive-sense RNA encoding a large polyprotein precursor (8, 9). The polyprotein is further cleaved into four structural proteins (VP1 to VP4) to form the viral capsid and seven nonstructural proteins (2A to 3D) for virus replication via the 2A protease and 3C protease (10, 11). Except for the cleavage of VP1/2A by the 2A protease (12) and the RNA-dependent cleavage of VP2/4 (13), the 3C protease is absolutely required for the cleavage of other junction sites within the polyprotein (14-16). Meanwhile, EV71 3C reportedly interferes with the polyadenylation of host cell RNA by digesting CstF-64, a critical host factor for 3= pre-mRNA processing, suggesting a novel mechanism by which picornaviruses utilize 3Cpro to impair host cell function (17). In addition, the 3C protease can also cleave numerous factors and regulators associated with cellular DNA-dependent RNA polymerases I, II, and III, such as the octamer-binding protein (OCT-1), TATA box-binding protein (TBP), cyclic AMP-responsive element-binding protein (CREB), transcription activator p53, histone H3, and DNA polymerase III (18-21). The pivotal role of 3C protease in EV71 replication makes it an attractive target for antiviral discovery (22).The crystal structure of unliganded EV71 3C protease showed that EV71 3C protease folded into two domains that are related to other picornaviral 3C protease structures (23). The complex structures of EV71 mutants H133G, E71A, E71D with the inhibitor rupintrivir are similar to that of the unliganded protease structure (24). Lu et al. thoroughly characterized the 3C proteases from EV71 and CVA16 and reported a series of structures of both enzymes in free, peptide-bound, or inhibitor-bound form (25). These findings provided precise molecular insights into the substrate recognition and inhibition of 3C protease.