(A) cDNA-transfected L929 cells were inoculated with high-titer EV-A71 (TCID₅₀/ml = 190,000). Forty-eight hours after inoculation, the cell lysates were harvested for Western blot analyses using specific antibodies. Mock-transfected L929 and EV-A71–infected RD cells were included as positive and negative controls, respectively. (B) Immunoprecipitation (IP) of hWARS from RD or hWARS-transfected L929 cells followed by pulldown of EV-A71. Endogenous hWARS from RD cells (lane 2) and overexpressed hWARS from hWARS-transfected L929 cells (lane 4) were first separately immunoprecipitated by anti-hWARS antibodies. The complexes were then inoculated with EV-A71 overnight at 4^oC. After washing away unbound viruses, the complexes were dissociated by anti–EV-A71 antibodies for Western blot (WB) analyses. Asterisks indicate nonspecific bands. (C) Pulldown of EV-A71 clinical isolates by recombinant hWARS protein. Three different clinical EV-A71 isolates were inoculated with recombinant hWARS protein immunocomplexes and detected using Western blot analyses as described in B. (D) Anti-hWARS antibody blockage of EV-A71 infection. Surface hWARS on RD cells was blocked with anti-hWARS antibodies for 1 hour before EV-A71 infection. Virus production in conditioned supernatants and virus protein expression in infected cell lysates are shown. (E) Saturation of EV-A71 virions by recombinant hWARS protein (ranging from 18.8 to 75.2 nM). EV-A71 was preincubated with recombinant hWARS protein before challenging the RD cells. Virus production and viral protein expression were measured as described in D. (F) Western blot analyses of EV-A71, hWARS, and hSCARB2 proteins in RD CRISPR/Cas9 (CRISPR) cell clones. Endogenous γ-tubulin was used as a loading control. The r value represents the Pearson’s correlation coefficient. Data in A–G are representative of 3 independent experiments, and data in D and E represent the mean ± SEM.