LAUSR

Significance Receptor usage can affect cell tropism and viral pathogenicity. CVB causes viral-induced heart disease, aseptic meningitis, and many other severe diseases globally that can engage CAR in host cells and selectively utilizes CD55 to infect them. However, the mechanism of CVB differential receptor usage and its dynamic entry into cells remains poorly understood. This study elucidates the molecular mechanism of VP3-234 residues as critical population selection determinants influencing CD55 affinity/specificity for naturally occurring CVB3 strains. Moreover, the demonstration that CAR facilitates viral uncoating with strain-dependent pH preferences suggests that the entry and infection of different enterovirus strains vary with the extent of pH dependence. Altogether, these findings expand our understanding of nonenveloped virus infections and provide clues for therapeutic interventions. Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.