Experiments were performed with EV71 (1095) or E11 (Gregory) that were expanded as described previously (39). In some cases, experiments were performed with light-sensitive NR viral particles, which were generated as described previously (21). Briefly, EV71 was propagated in the presence of NR (10 μg/ml) in the semi-dark and was subsequently purified in semi-dark conditions by ultracentrifugation over a sucrose cushion, as described (39).

For infections, wells were infected with 106 plaque-forming units (PFU) of the indicated virus. Virus was preadsorbed to the apical or basolateral surfaces for 1 hour at room temperature (basolateral infections were initiated by inverting the Transwell inserts). Infections were then initiated by shifting to 37°C and allowed to proceed for the times indicated. For NR virus experiments, particles were exposed to light (on a light box) for 20 min at 6 hours p.i. and then infected for the indicated number of hours after light exposure. In some cases, cells were exposed immediately following adsorption (0 hours), which served as a control. E11 and EV71 plaque assays were performed in HeLa cells overlayed with 1.0 or 0.8% agarose, respectively; plaques were enumerated following crystal violet staining.

Binding assays were performed by preadsorbing 106 PFU of the indicated virus to the apical or basolateral surfaces for 60 min at room temperature, followed by extensive washing with 1× phosphate-buffered saline (PBS). Following washing, RNA was isolated immediately, and RT-qPCR was performed, as described below.

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