Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in
hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput
small interfering RNA (
siRNA) screening and validation, we found that the
infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by
caveolin,
clathrin, and macropinocytosis but dependent on
ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (
GTP)-binding protein of the Ras superfamily. The specific
siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by
NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with
siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71
viral protein. Furthermore, ARF1, another small
GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that
NAV-2729 significantly alleviated mortality caused by EV71
infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for
drug development.