Ev71 3 C Protease And Its Interaction With Antiviral Drugs Of Structural Biology Research

Human enterovirus71(EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects children and infants. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date.The genome of EV71consists of a positive sense, single-stranded RNA of7400bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV713C protease (3Cpro). Previous studies showed that EV713Cpro can diggest CstF-64during the infection, thus, impair the mRNA metabilism of the host cell. Recently, MAVS and TRIF were found cleaved by EV713Cpro, leading to the attenuated host type I interferon signaling via RIG-I like helicases pathway. EV713C also possesses RNA binding activities, suggesting its role in RNA genome replication. In summary, EV713C is capable of performing multiple tasks in many key events in viral replication and pathogen-host interactions. The central roles played by EV713Cpro make it an appealing target for antiviral drug development.In this study, we determined the first crystal structure of EV713Cpro and analyzed its enzymatic activity. The crystal structure shows that EV713Cpro has a typical chymotrypsin-like fold that is common in picornaviral3Cpro. Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a previously unobserved novel open conformation in EV713Cpro. We identified two important residues located at the base of the β-ribbon, Gly123and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the mobilities of the hinge residues are important to EV713Cpro proteolytic activities. Rupintrivir is an HRV3Cpro inhibitor designed to combat common cold caused by rhinovirus infections. Interestingly, there are increasing evidences showing that AG7088is also effective against enteroviruses. We find that rupintrivir is a potent inhibitor against EV71and the drug can inhibit the protease activity of EV713Cpro in vitro. In order to elucidate the structural basis of the inhibition, we determined high-resolution structures of EV713Cpro/rupintrivir complex providing the novel molecular insights to the substrate recognition of the protease. These structures demostrate that the binding mode of rupintrivir to EV713Cpro in general is simialr to that of rupintrivir/HRV3Cpro, however, some differences are also found. The C-terminal ethyl easter of rupintrivir doesn’t accommodate in the leavding group pockets of EV713Cpro. EV713Cpro possesses a surface ressesive S2’substrate specificity poket. The similar feature is present in CVB3Cpro and PV3Cpro, but not in HRV3Cpro. Our structural and enzymic studies of EV713Cpro mutants demonstrate the irreplaceable role of the catalytic Glu71, spportint the notion that this family of proteases adpots the catalytic triad mechanism. In addtion, we found the role of a previously uncharacterized residue Arg39that is important to proteolysis. Our findings add the novel details to the catalytic mechanism of viral3C protease. In summary, our data provides a framework for structure-guided inhibitor design against EV713Cpro.