Effect of ammonium chloride on the reovirus egress machinery. HBMECs were either mock infected or adsorbed with reovirus strains T3D and T1L M1-P208S at an MOI of 1 PFU/cell, treated at 24 hpi with 20 mM ammonium chloride (NH₄Cl), and incubated for an additional 24 h. Cells were fixed and processed for confocal microscopy and EM. (A) Confocal microscopy of infected cells incubated in the absence or presence of NH₄Cl and stained with mouse anti-σ1 monoclonal antibody 9GB5 (green). Lysosomes are labeled with anti-LAMP-1 (red). In untreated T3D-infected cells (image on the left), lysosomes with virions (arrows) surround VIs (asterisks). In the perinuclear region of infected cells treated with NH₄Cl (image on the right), lysosomes near VIs do not contain virions (arrows), and reovirus σ1 protein concentrates in perinuclear puncta (arrowheads). Scale bars, 25 µm. (B) HBMECs were adsorbed with reovirus T1L-M1-P208S at an MOI of 1 PFU/cell and either untreated or treated with NH₄Cl. Titers of extracellular (supernatant) and intracellular (cell lysate) virus were determined by plaque assay. The results are presented as mean ± SEM of three independent experiments, each with two technical replicates. *, P < 0.05, unpaired two-tailed Student’s t test. (C and D) EM of T1L-M1-P208S-infected cells incubated with or without NH₄Cl. (C) In untreated infected cells, modified lysosomes (SOs) near VIs contain mature virions (arrows). In NH₄Cl-treated cells, SO-like structures without virions are observed near VIs. (D) MCs with mature virions are observed in the cytoplasm of untreated infected cells. MCs filled with virions are not detected in cells treated with NH₄Cl. N, nucleus. Scale bars, 500 nm.