Clathrin‐Independent Carriers/Glycosylphosphatidylinositol‐Anchored‐Protein‐Enriched Endosomal Compartment Endocytic Pathway Is Critical for Enterovirus A71 Entry Into Human Oral Epidermoid Carcinoma KB Cells

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease, which can lead to further infections and spreads via the oral cavity; however, the mechanism of how EV-A71 infects the human oral cavity remains unclear. Screening and validation using small-interfering RNAs and chemical inhibitors showed that EV-A71 entry into human oral epidermoid carcinoma KB cells was independent of clathrin-, caveolin-, endophilin-, dynamin-, and macropinocytosis-mediated pathways. However, the clathrin-independent carriers/GPI-anchored-protein-enriched endosomal compartment (CLIC/GEEC) pathway is crucial for EV-A71 entry into KB cells and normal human oral epithelial cells (NHOEC), which requires the entire actin cytoskeleton and membrane cholesterol. Inhibition of GBF1, ARF1, CDC42, PICK1, GRAF1, and IRSp53, the key molecules of the CLIC/GEEC pathway, significantly suppressed EV-A71 entry and infection. Confocal microscopy showed that internalized EV-A71 colocalized with CDC42 and GRAF1. Knockdown of CDC42 or GRAF1 reduced the number of internalized viral particles, which were predominantly localized at the plasma membrane. By using the sucrose density gradient centrifugation, EV-A71 and glycosylphosphatidylinositol-anchored GFP (GPI-GFP) were observed within the same low-density components. Furthermore, endocytosed EV-A71 was colocalized with GRAF1 and GPI-GFP and transported to the late endosomes. Mouse experiments demonstrated that the GBF1 inhibitor, Golgicide A, significantly reduced EV-A71 infection-induced mortality. Immunohistochemical staining and histopathological section analysis revealed that Golgicide A markedly decreased the viral load in brain tissue and oral epithelium, and alleviated the pathological damage induced by the virus in brain tissue. Our findings reveal a novel pathway for EV-A71 entry into KB cells and provide a new target for drug development.