CHO cells were either mock transfected or transiently transfected with plasmid encoding NgR1 or JAM-A for 48 h. (A) Transfected cells were treated with PI-PLC, which cleaves GPI-anchored proteins, for 1 h prior to adsorption with reovirus T3SA- at an MOI of 10 PFU/cell. (B) Reovirus T3SA- was incubated with soluble Fc-tagged NgR1, JAM-A, or CAR at the indicated concentrations prior to inoculation of transfected cells at an MOI of 10 PFU/cell. (A and B) Cells were fixed at 20 h and scored for reovirus antigen using indirect immunofluorescence. Results are expressed as the mean FFU/field for three fields of view in triplicate samples. Error bars indicate SD. **, P < 0.005; ***, P < 0.0005 (as determined by Student’s t test in comparison to NgR1- or JAM-A-transfected cells). See also Figure S4.