EV-D68 exploits clathrin-mediated endocytosis and compensatory macropinocytosis for cellular entry

Despite growing health concerns about enterovirus D68 (EV-D68), its cellular entry mechanisms remain incompletely understood. In this study, we investigated the endocytic pathways exploited by the sialic acid-dependent EV-D68 TW-02795-2014 strain using pharmacological inhibitors, siRNA-mediated knockdown, dominant-negative constructs, and electron microscopic analysis. Chemical inhibition of clathrin-mediated endocytosis (CME), macropinocytosis, endosomal acidification, and other endocytic pathways reduced viral entry and protein expression, suggesting the involvement of multiple routes in viral entry. Co-localization of EV-D68 with clathrin and the effects of CME inhibitors supported CME as a major entry route. However, siRNA knockdown of CME components in RD cells inhibited the cellular uptake of transferrin, yet had no significant impact on EV-D68 infection. This was also seen in A549 cells. Notably, CME disruption by clathrin knockdown led to Rac1 upregulation and increased macropinocytosis, which facilitated EV-D68 entry. Transmission electron microscopy revealed membrane-associated EV-D68 particles organized in clusters underneath the cell surface during infection, potentially undergoing bulk internalization. In contrast, purified virions lacking membranous material strictly required CME for successful infection. These findings reveal a dual entry strategy: single (membrane-free) EV-D68 particles utilize CME, while membrane-associated virions access host cells via macropinocytosis, particularly under conditions of CME inhibition. This study highlights the complexity of EV-D68 endocytosis and underscores the importance of compensatory mechanisms in viral uptake.IMPORTANCEUnderstanding how EV-D68 enters host cells is crucial for developing antiviral strategies. This study uncovers a dual-entry mechanism used by a sialic acid-dependent EV-D68 strain: clathrin-mediated endocytosis (CME) for single virus particles, and macropinocytosis as an alternative route for membrane-associated particles or under conditions where CME is impaired. Notably, disruption of CME induces a compensatory upregulation of macropinocytosis, mediated by increased Rac1. These findings challenge prior assumptions of a singular viral entry pathway and emphasize the need to consider endocytic plasticity when designing antiviral interventions targeting EV-D68 or similar viruses.