Cryo-EM Imaging of RSV Infection

For cryo-EM/ET infection studies, HeLa cells were grown on gold Quantifoil R 2/1 grids (Quantifoil, Großlöbichau, Germany) and inoculated with RSV A2-mK⁺ at a M.O.I. of 10. The infected cells were rocked for 1 h at room temperature to facilitate virus binding and were then washed twice with PBS to remove the unbound viral particles. New medium containing 600 nM fusion inhibitor (BMS-433771) [51 (link),52 (link)] was added to prevent viral entry and fusion events, and the infected cells with fusion inhibitor were allowed to incubate for 24 h. The samples were processed for cryo-EM/ET imaging as described below.