Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

Abstract

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

NLRP3 deficiency exacerbates enterovirus infection in mice

“…As in the example of enterovirus infection, EV-A71-encoded proteinases 2A and 3C were shown to directly cleave NLRP3 (19). In addition, it was found that EV-A71 inhibits pyroptosis via virus proteinase 3C-mediated cleavage of gasdermin D (44). Similarly, in the present study, we discovered that protein levels of NLRP3 are reduced after CVB3 infection as a consequence of CVB3 proteinaseinduced cleavage and subsequent degradation by cellular proteases.…”

Mechanism and Regulation of Gasdermin-Mediated Cell Death

“…The 3C protease of EV71 cleaves GSDMD at an amino-terminal site, inhibiting GSDMD ( Fig. 3C; Lei et al 2017). EV71 infection resulted in this GSDMD cleavage in multiple cell lines (Lei et al 2017).…”

NLRP3 Inflammasome—A Key Player in Antiviral Responses

“…A couple of viruses can modulate the effector functions of the NLRP3 inflammasome. The EV71 protease 3C cuts the NLRP3 inflammasome activation effect factor GSDMD, at 193-194 residues, instead of 275-276 residues by caspase-1 (74). This aberrantly cleaved GSDMD product fails to induce cell pyroptosis of the infected cell and, as a result, promote viral replication and attains the objective of viral evasion.…”