Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

Wang, Jing; Fan, Ting-Jun; Xue, Yuan; Wú, Zhìqiáng; Guo, Li; Lei, Xiaobo; Wang, Jianwei; Wang, Meitian; Jin, Qi; Cui, Sheng

doi:10.1128/jvi.05107-11

Cited by 80 publications

(114 citation statements)

References 24 publications

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“…5C). In contrast, this leaving-group side pocket in HRV 3C pro is partially occupied by the ethyl ester of AG7088 (22). Because the P1= position is replaced by an aldehyde group in NK-1.8k or a cyanohydrin group in compound 9, the structural finding that the leaving-group side pocket is in a free state is not surprising (Fig.…”

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confidence: 79%

“…In the structures of EV71 3C pro in complex with NK-1.8k, this ␤-ribbon changes to a closed conformation and seals the bottom of the substrate-binding groove (Fig. 2B), which is similar to the conformation when binding substrate or inhibitors (22,23).…”

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confidence: 92%

“…5). H40 is a key residue that not only participates in the catalytic reaction but also structurally forms the ceiling of the S2 site by its imidazole group (22). In the structures of the WT or N69S EV71 3C pro complex with NK-1.8k and compound 9, the side chain of H40 maintains the same orientation that it maintains in natural substrate-bound or unliganded EV71 3C pro .…”

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confidence: 99%

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Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor

Wang

Cao

Zhai

et al. 2017

Antimicrob Agents Chemother

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Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3C pro ). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3C pro . Comparison of these structures with the structure of unliganded EV71 3C pro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3C pro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1= to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses.KEYWORDS EV71, protease, inhibitor, crystal structure, mechanism H and-foot-and-mouth disease (HFMD) is a common viral illness among infants and young children that causes fever, sore throat, blisters, pharyngitis, mouth ulcers, and a rash on the hands and feet (1, 2). Enterovirus 71 (EV71) has been the main causative agent of HFMD in recent years. Unfortunately, no drugs for the treatment of this disease are available. EV71 is classified as a member of Enterovirus species A within the genus Enterovirus of the Picornaviridae family (3). In the virus replication cycle, the 7.4-kb positive-sense, single-stranded RNA genome of EV71 is translated into a polyprotein which is subdivided into three primary precursors, P1, P2, and P3. Four structural proteins, VP1 to VP4, derived from the P1 portion, form the capsid of the mature virion (4), and the P2 and P3 regions are cleaved by viral proteases, forming seven individual nonstructural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D (5, 6). The correct replication of EV71 is dependent on the effective cleavage of the viral polyprotein by viral 2A protease (2A pro ) and 3C protease (3C pro ) (7). Of these two proteases, 3C pro not only takes major responsibility for polyprotein cleavage, with the exception that 2A pro is responsible for the cleavage of VP1/2A and 3C/3D, but also plays multiple roles in various biological processes (8).EV71 3C pro is a classical cysteine protease. Cysteine proteases are a large family containing many members functioning in various physiological processes (9). The

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confidence: 79%

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confidence: 92%

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confidence: 99%

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Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor

Wang

Cao

Zhai

et al. 2017

Antimicrob Agents Chemother

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“…To explore whether the protease activity of EV71 3C is required for the cleavage of GSDMD, we evaluated the impact of rupintrivir, which is an inhibitor of 3C protease with a broad spectrum of activity against picornaviruses (37)(38)(39). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In order to further verify this issue, we carried out mutational analysis. His40, Glu71, and Cys147 are key amino acids which form the catalytic triad of EV71 3C (39). H40D and C147S substitutions in the active site of EV71 3C disrupt the protease activity.…”

Section: Resultsmentioning

confidence: 99%

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

Lei

Zhang

Xiao

et al. 2017

J Virol

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120

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Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD ). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD ). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response.IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1␤ (IL-1␤) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD 1-275 (p30) inhibits EV71 replication whereas GSDMD 1-193 does not. These results reveal a new strategy for EV71 to evade the antiviral response. KEYWORDS EV71, HFMD, 3C, GSDMDE nterovirus 71 (EV71) is a major etiological agent of hand-foot-and-mouth disease (HFMD) which affects infants and children younger than 5 years of age. Most of these infections are self-limited with mild symptoms, including fever and vesicular eruptions mainly on the skin of hands and feet and in the mouth. However, some patients show severe neurological diseases, including aseptic meningitis, acute flaccid paralysis, encephalitis, and pulmonary edema. Since it was first isolated in California in 1974 (1-3), EV71 infection has caused many large-scale epidemics in the world, especially in the Asia-Pacific region (1, 4-6). To date, there are no effective therapeutic drugs for EV71 infection.EV71 belongs to the Enterovirus genus of the family Picornaviridae. The viral genome is approximately 7,500 nucleotides in length, with a single open reading frame that encodes a large precursor protein that is subsequently processed into three structural (VP0, VP1, and VP3) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins by the virus-encoded protease 2A or 3C (7). The three structural proteins and the RNA form provirions, in which VP0 is cleaved into VP2 and VP4 by RNA or 3CD, but the

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RNA‐virus proteases counteracting host innate immunity

Lei

Hilgenfeld

2017

FEBS Letters

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Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen‐associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern‐recognition receptors of the host, and innate immune responses are induced. Through production of type‐I and type‐III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive‐sense single‐stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses.

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Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

Cited by 80 publications

References 24 publications

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

RNA‐virus proteases counteracting host innate immunity

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