PGN-ZA inhibits intracellular trafficking of endocytosed viruses. (A) Influenza virus was labeled with Alexa 647 succinimidyl ester (green) and mixed with PGN-ZA. The mixture was then added to MDCK cells at 4 °C for 1 h and washed away with PBS. Medium containing Lysotracker (red) and either PBS or PGN-ZA were added to the samples and were immediately moved to a 37 °C. Samples were taken at time points 0, 5, 15, 30, and 60 min, and cells were fixed and stained with E-cadherin (magenta) and DAPI, and imaged by fluorescent microscopy at 60× magnification. For each sample, the left panel shows E-cadherin (cell boundary) and DAPI (nuclei) staining and the right panel shows viruses, Lysotracker (acidic compartments) and DAPI staining. Representative images are shown. (B) The mean ± SEM of the number of viral particles per cell was quantified from the microscopy images and normalized to the PBS control. (C) Influenza virus was incubated at pH 5 or pH 7 (as a control) in the presence or absence of PGN-ZA for 15 min at 37 °C. The level of infectious viruses remaining after the treatment was quantified by serial dilution and the plaque assay. Two concentrations of PGN-ZA were used. The mean ± SD of infectious viruses shown here are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.