Visualizing reovirus entry and egress in HBMECs using confocal microscopy and EM. (A) HBMECs were adsorbed with reovirus T1L M1-P208S at an MOI of 20 PFUs/cell and imaged by confocal immunofluorescence microscopy without cell permeabilization at 4 hpi. Virus (green) was labeled with a reovirus-specific antiserum, actin (red) was labeled with phalloidin, and WGA (blue) was used to label the plasma membrane. Nuclei are labeled in gray. Reovirus particles are observed in a punctate pattern on the cell surface. Scale bars, 10 µm. (B) EM of ultrathin sections of infected cells processed by high-pressure freezing and freeze substitution. The images show reovirus attachment and internalization into organelles compatible with endosomes. Scale bars, 100 nm. (C) Reovirus ISVPs were prepared by digestion of purified virions with chymotrypsin. (D) SDS-PAGE shows that ISVPs lack the σ3 capsid protein and contain the δ cleavage product of μ1C protein. (E) Viral titers following adsorption of HBMECs with reovirus ISVPs at an MOI of 1 PFU/cell. (F) Cells were adsorbed with reovirus ISVPs, fixed 18 hpi, permeabilized, and imaged by confocal immunofluorescence microscopy following staining with anti-σ3 mouse monoclonal antibody 10C1. Fluorescent signal concentrates in discrete zones at the basal surface are shown in the confocal total projection and lateral merged image. Scale bar, 10 µm. (G) EM of basal surfaces of infected HBMECs adsorbed with ISVPs shows reovirus egress zones. Arrowheads point to virion-containing MCs, which are close to plasma membrane (pm) and releasing virions (arrows). Representative images from 100 cells obtained in six independent experiments are shown. Scale bars, 200 nm.