FIG 6.

ZCL278 prevents the release of JUNV RNP into the cytosol and GP-mediated fusion. (A to C) JUNV material released into the cytosol, measured using immunofluorescence labeling. (A) (Left) NP⁺ and GP⁺ objects that are considered to be JUNV particles with an intact envelope within endosomes; (right) NP⁺ and GP⁻ JUNV material released from endosomes. (B, C) To detect both states, Vero cells were left untreated or treated with increasing amounts of ZCL278 and incubated with JUNV-A647 for 30 min at 37°C. Cells were stained with two noncompetitive anti-NP antibodies coupled to different fluorophores (NP-A488 and NP-A568). (B) Representative snapshots of the inverted fluorescence intensity for each individual channel. Yellow arrowheads, objects positive for all three fluorophores and thus considered to be NP⁺ and GP⁺ particles; green arrowheads, NP⁺ and GP⁻ objects considered to be released events; all detected objects were classified as either NP⁺ and GP⁺ or NP⁺ and GP⁻; red arrowhead, a GP⁺ and NP⁻ object (not taken into account in the following analysis) that possibly represents an endosome with which fusion already occurred. (C) Quantification of the percentage of JUNV material (NP⁺ and GP⁻) released from endosomes. The data are the averages ± SDs from two or three experiments, with each experiment corresponding to 10 fields of view with more than 100 objects counted per condition. Statistical analysis using the two-tailed Student t test was used to show significance. *, P < 0.05; **, P < 0.005. (D) Viral fusion at the plasma membrane was artificially induced by placing Vero cells on which JUNV virions were prebound at 4°C, followed by dropping the pH of the extracellular medium in the presence or absence of 50 μM ZCL278. The histogram represents the percentage of infected cells for the different conditions measured by flow cytometry. The data correspond to the means ± SDs from duplicate experiments with at least 10,000 cells per condition, and the histogram is representative of the data from two independent experiments. Statistical analysis for significance was performed using the two-tailed Student t test. *, P < 0.05; **, P < 0.005.