Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

Abstract

The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry.

Fig. 1

Endocytosis of EB2 in different cell lines. EB2-expressing cells (Vero, MDCK, PAEC, and HeLa) were incubated with EphB4/Fc for 1 h at 37 °C to allow binding and endocytosis to proceed. Surface-remained EphB4/Fc was stained with a rhodamine-conjugated anti-human IgG antibody (surface). After fixation and permeabilization, internalized EphB4/Fc was detected by a FITC-conjugated secondary antibody (intracellular).

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 G_tag-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.

Fig. 3

Effect of endocytosis inhibitors, NH₄Cl, and cathepsin inhibitors on F protein cleavage. Vero cells expressing the NiV F protein were radiolabeled with [³⁵S]-Promix for 10 min and were then incubated in chase medium containing the indicated inhibitors for 2 h. F proteins were immunoprecipitated from cell lysates, separated on a 12% SDS gel under reducing conditions, and subjected to autoradiography.

Fig. 4

Effect of inhibitor treatments during and after virus entry on NiV infection. (A) Inhibitor treatment before and during virus entry. Vero cells were infected with NiV at a MOI of 0.2. Cells were treated with the indicated inhibitors for 1 h prior infection and for 1 h at 37 °C during virus adsorption and entry, or were left untreated (control). Then, inhibitors and input virus were removed, cells were washed and further incubated in medium for 24 h at 37 °C. After fixation and permeabilization, cells were incubated for 1 h on ice with a polyclonal anti-NiV serum and primary antibodies were detected with rhodamine-conjugated secondary antibodies. (B) Inhibitor treatment after virus entry. After infection of Vero cells with NiV for 1 h at 37 °C, cells were washed and then incubated in the absence (control) or presence of the indicated inhibitors. At 24 h p.i., immunostaining was performed as described above. There was no generalized toxicity effect of any of the inhibitors at the concentrations used in this experiment.

Fig. 5

Effect of sucrose and NH₄Cl treatment on influenza virus entry. Vero cells were infected with FPV at a MOI of 0.01. Cells were either left untreated (control) or incubated with the indicated inhibitors for 1 h prior infection and for 1 h during virus adsorption and entry. After washings to remove the inhibitors and input virus, residual virus attached to the cell surface was inactivated by acidic pH treatment. At 16 h p.i., cells were fixed and stained with a FPV-specific monoclonal antibody. Primary antibodies were detected with rhodamine-conjugated secondary antibodies and nuclei were visualized by DAPI staining.

Fig. 6

F protein cleavage in purified NiV. Mock-sample and purified NiV were subjected to SDS-PAGE under reducing conditions and analyzed by Western blotting. Viral proteins were either detected with a polyclonal anti-NiV serum (anti-NiV) or with NiV F-specific antibodies (anti-F).