EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

Abstract

Activation of NLRP3 inflammasome is important for effective host defense against invading pathogen. Together with apoptosis-associated speck-like protein containing CARD domain (ASC), NLRP3 induces the cleavage of caspase-1 to facilitate the maturation of interleukin-1beta (IL-1β), an important pro-inflammatory cytokine. IL-1β subsequently plays critical roles in inflammatory responses by activating immune cells and inducing many secondary pro-inflammatory cytokines. Although the role of NLRP3 inflammasome in immune response is well defined, the mechanism underlying its assembly modulated by pathogen infection remains largely unknown. Here, we identified a novel mechanism by which enterovirus 71 (EV71) facilitates the assembly of NLRP3 inflammasome. Our results show that EV71 induces production and secretion of IL-1β in macrophages and peripheral blood mononuclear cells (PBMCs) through activation of NLRP3 inflammasome. EV71 replication and protein synthesis are required for NLRP3-mediated activation of IL-1β. Interestingly, EV71 3D protein, a RNA-dependent RNA polymerase (RdRp) was found to stimulate the activation of NLRP3 inflammasome, the cleavage of pro-caspase-1, and the release of IL-1β through direct binding to NLRP3. More importantly, 3D interacts with NLRP3 to facilitate the assembly of inflammasome complex by forming a 3D-NLRP3-ASC ring-like structure, resulting in the activation of IL-1β. These findings demonstrate a new role of 3D as an important player in the activation of inflammatory response, and identify a novel mechanism underlying the modulation of inflammasome assembly and function induced by pathogen invasion.

Author Summary

The immune system protects the infected host and clears the invading pathogens. An important part of the innate immune response is the activation of NLRP3 inflammasome, which is induced upon exposure to pathogens. Activated inflammasome subsequently regulates the maturation of IL-1β that plays an important role in inflammatory response. Enterovirus 71 (EV71) is a highly contagious virus causing hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children by inducing many pro-inflammatory cytokines. Although NLRP3 inflammasome plays important role in regulating host immunity and viral infection, the assembly of NLRP3 inflammasome induced by viral infection is not known. In this study, we demonstrate that EV71 3D RNA polymerase activates NLRP3 inflammasome by binding to NLRP3. More importantly, 3D was found to interact with NLRP3 to facilitate the assembly of inflammasome complex by forming a specific ring-like structure. Therefore, these findings demonstrate a new role of viral 3D polymerase in the activation of inflammatory response, and identify a novel mechanism underlying the regulation of inflammasome assembly in responding to pathogen infection, which would provide insights into the prevention and treatment of viral infection.