KREMEN1 residue W94 is essential for receptor binding and infection by a major group of enteroviruses

KREMEN1 (KRM1) serves as a cellular receptor for a major group of enteroviruses causing hand, foot, and mouth disease (HFMD), including CVA2-CVA6, CVA8, CVA10, and CVA12. The viral VP2 residue K140 has recently been identified as completely conserved among these viruses and critical for interaction with KRM1 and viral infection. However, the molecular determinants on the receptor side that govern broad enterovirus recognition remain incompletely understood. Here, we identify KRM1 tryptophan 94 (W94) as an essential residue for KRM1-mediated enterovirus infection. Alanine scanning of 29 KRM1 residues at the CVA10-KRM1 interface revealed that only W94A nearly abolished CVA10 infection. Structural analysis revealed that KRM1 W94 engages in a π-cation interaction with the viral VP2 residue K140, which is critical for receptor binding. Unlike wild-type mice, which succumbed to CVA10 infection, CRISPR-engineered mouse Krm1 W94A mutant mice were fully resistant. Moreover, the W94A mutation conferred universal resistance to all KRM1-dependent enteroviruses (CVA2-CVA6, CVA8, CVA10, and CVA12), both in cell culture and in vivo, indicating that the W94 residue is essential for infection by these viruses. Collectively, these findings establish KRM1 W94 as a critical receptor determinant that pairs with the universally conserved VP2 K140 to enable enterovirus infection. This work provides a structural and mechanistic basis for the broad recognition of KRM1-dependent enteroviruses and offers insights that may guide the future development of broad-spectrum antiviral strategies.IMPORTANCEHand, foot, and mouth disease affects millions of children worldwide each year; however, no effective antiviral drugs are available. Many causative viruses, including coxsackievirus A10 and A6, rely on the cellular receptor KREMEN1 to enter human cells. However, which parts of the receptor are critical for viral binding and infection have remained unclear. Here, we identified tryptophan 94 (W94) of KREMEN1 as critical for viral infection. Introducing the W94A mutation abolished viral binding and infection. Structural analysis revealed that W94 interacts directly with a completely conserved residue K140 on the viral capsid protein VP2. Notably, mice carrying this mutation were completely resistant to all KREMEN1-dependent viruses. These findings provide a molecular basis for virus-receptor recognition and highlight a promising target for broad-spectrum therapies against hand, foot, and mouth disease.