Fig. 2.
VLP- and virus-uptake assays. (A) Vero cells were incubated for 1 h at 4 °C (control-4 °C) or at 37 °C (control-37 °C) with purified NiV Gtag-containing VLPs in combination with a monoclonal antibody directed against the HA-tag. Surface-bound VLPs were visualized by incubation with FITC-conjugated anti-mouse IgG antibodies at 4 °C. After permeabilization with methanol–acetone, intracellular VLPs were stained with a rhodamine-conjugated secondary antibody. Nuclei were visualized by DAPI staining. Merged pictures of the DAPI, FITC and rhodamine fluorescence channels are shown. (B) Cells were preincubated for 30 min at 37 °C with the following endocytosis inhibitors: 0.45 M sucrose (sucrose), 25 µM chlorpromazine (chlorpromazine) or 5 mM β-methyl-cyclodextrin (MCD). Incubation with VLPs and HA-tag antibody was performed for 1 h at 37 °C in the presence or absence of the inhibitors. Surface-bound and intracellular VLPs were stained as described above. (C) Vero cells were incubated with infectious NiV particles for 2 h at 4 °C. After incubation with anti-NiV serum for 30 min on ice, cells were either shifted to 37 °C or kept at 4 °C for 20 min. Surface-bound and intracellular antibodies were visualized as described above.