Nipah virus infection and glycoprotein targeting in endothelial cells

Abstract

Background: The highly pathogenic Nipah virus (NiV) causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB) and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells.

Results: As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar), not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting.

Conclusion: We conclude that the NiV glycoprotein distribution is responsible for lateral virus spread in both, epithelial and endothelial cell monolayers. However, the prerequisites for correct protein targeting differ markedly in the two polarized cell types.

Figure 1

NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.

Figure 2

Distribution of the NiV glycoproteins and the NiV receptor EB2 on the surface of polarized endothelial cells. PBMEC (A) and PAEC-EB2 (B and C) were cultured on filter supports for 6 or 5 days, respectively. (A, B) Polarized cell cultures were infected with NiV at a m.o.i. of 0.5. At 24 h p.i., cells were inactivated and fixed with 4% PFA and then incubated from both sides with monoclonal antibodies directed either against the F or the G protein, followed by incubation with AlexaFluor 568-conjugated secondary antibodies. Confocal horizontal (xy) sections through the apical part of the cell monolayer are shown in the left panel. White lines indicate the area along which vertical sections were recorded. Vertical (xz) sections through the foci are shown on the left panel. (C) Cells were fixed and surface-stained from both sides with a EB2-specific ligand (EphB4/Fc) and a AlexaFluor 568-labelled secondary antibody. Then cells were permeabilized and incubated with a VE-cadherin specific antibody and a AlexaFluor 488-conjugated secondary antibody. Confocal horizontal (xy) and vertical (xz) sections are shown.

Figure 3

Surface distribution of wild-type and mutant F and G proteins. (A) Amino acid sequences of the cytoplasmic domains of wild-type and mutant F and G proteins. Numbers above the sequences indicate amino acid positions. Boldface letters indicate exchanged amino acid residues. Vertical lines indicate the beginning of the predicted transmembrane domains. (B and C) Surface distribution of wild-type F and G proteins in polarized endothelial cells. PAEC stably expressing either wild-type or mutant NiV F (B) or G (C) were grown on filter supports for 5 days and then incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with AlexaFluor 568-conjugated secondary antibodies. Confocal vertical sections through the cell monolayers are shown. (D) Cell surface proteins were labelled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin and chemiluminescence.