SPNS1 is an essential cellular factor for EV-A71 by acting as a transporter of viral pocket factor.

Human enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease. Cellular factors critical for EV-A71 infection remain enigmatic. Here, we performed CRISPR/Cas9 screens and identified sphingolipid transporter 1 (SPNS1) as an essential factor for EV-A71. SPNS1 deficiency inhibits infection of EV-A71 and 9 of 11 examined enteroviruses. Mechanistically, the endo/lysosomal localization of SPNS1 and the acidification of the endo/lysosomes are essential for SPNS1 to support EV-A71 infection. SPNS1 deficiency inhibits EV-A71 genomic RNA replication, but barely affects replication of EV-A71 RNA directly transfected into the cytoplasm. SPNS1 interacts with the EV-A71 capsid protein VP1 and entry receptor SCARB2 in the endo/lysosomes, where it acts as a transporter to release the viral pocket factor into the cytosol, leading to uncoating. Animal experiments show that SPNS1 deficiency results in reduced viral loads, pathological effects, and lethality following EV-A71 infection. Our findings collectively identified SPNS1 as a transporter of the EV-A71 viral pocket factor.