Fig. 8: Heparan sulfate serves as the primary attachment receptor for CVA6, whereas KRM1 functions as a post-attachment entry receptor.

a Electrostatic surface potential maps of CVA6-HeB viral pentamers. Color scheme: red for negative potential, white for neutral, and blue for positive potential. The black dashed circle highlights the positively charged patch. b Schematic diagram illustrating the sulfation process of heparan sulfate and its role in mediating cellular attachment of multiple enteroviruses (EV). c Wild-type RD cells, ΔKRM1 cells, and ΔSLC35B2 cells were infected with CVA6-HeB, and viral titers were quantified at 48 hpi. Data are means ± SD of triplicate biological samples. p = 0.0422 (ΔKRM1), p = 0.045 (ΔSLC35B2). d CVA6 specifically binds heparin-agarose beads. CVA6-HeB supernatant was loaded onto heparin or control columns, followed by washing and elution. Samples were analyzed by western blotting with anti-CVA6-VP0 antibody. FT, flow through. Two independent experiments were performed, with similar results. e Inhibition of CVA6 attachment to RD cells by soluble heparin. CVA6-HeB was treated with heparin and allowed to attach to cells for 2 h at 4 °C. Attached virus was quantified by RT-qPCR analysis and normalized to β-actin. Data are means ± SD (n = 4 for virus-only; n = 3 for others). p = 0.2084 (heparin-50 μg/ml), p = 0.0004 (heparin-500 μg/ml). f Viral attachment assay. CVA6-HeB was incubated with wild-type RD cells, ΔKRM1 cells, and ΔSLC35B2 cells at 4 °C for 2 h. After washing, cell-bound virus was quantified via RT-qPCR. Data are means ± SD of four biological replicates. p = 0.2859 (ΔKRM1). g Viral internalization assay. After virus binding at 4 °C, cells were shifted to 37 °C. Internalized virus was quantified by RT-qPCR following trypsin removal of surface virions. Data are means ± SD of four biological replicates. Source data are provided as a Source Data file. Statistical note for (c, e, f, g): significance was determined by two-tailed t-test. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. h Two-step model of CVA6 entry: attachment via heparan sulfate, followed by KRM1-mediated internalization.