FIG 7.

GERV relies on acid-activated membrane fusion for infection. (A to D) Cells were pretreated with endosomal pH-disrupting drugs at the indicated concentrations and were then infected with GERV at an MOI of 5 in the continuous presence of chloroquine (A), NH₄Cl (B), bafilomycin A1 (C), or concanamycin B (D). Infection was quantified by flow cytometry as described in Fig. 1A, and the data were normalized to those in control samples without inhibitor treatment. (E) GERV was bound at an MOI of 5 to confluent monolayers of A549 cells for 1 h on ice. Cells were then washed and treated at the indicated pH values at 37°C for 1.5 min. Infected cells were subsequently incubated overnight in the presence of NH₄Cl (50 mM) at pH ∼7.4 to block virus penetration from endosomes. Consequently, the release of viral genomes only from the plasma membrane was monitored in this assay. Infection was analyzed by flow cytometry and expressed as the percentage of infected cells relative to total cells. (F) R18-labeled GERV was bound at an MOI of 10 to A549 cells on ice before warming to 37°C for 1 h. The increase in the fluorescence signal corresponded to dequenching of the lipid dye R18 after virus fusion with endosomes in living cells and was measured with a fluorimeter. NH₄Cl was used to block virus fusion by raising the endosomal pH and, hence, to determine the fluorescence background due to spontaneous translocation of the R18 molecules between the viral envelope and the adjacent cell membrane. RU, relative unit. (G and H) Cells were pretreated with KCl (G) or TEA (H), a broad-spectrum K⁺ channel blocker. Cells were infected with GERV at an MOI of 5 in the continuous presence of the perturbants. The percentage of infected cells was quantified by flow cytometry as described in Fig. 1A, and the data were normalized to those in samples not treated with the perturbants.