Selective receptor expression restricts Nipah virus infection of endothelial cells

Abstract

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes severe diseases in animals and humans. Endothelial cell (EC) infection is an established hallmark of NiV infection in vivo. Despite systemic virus spread via the vascular system, EC in brain and lung are preferentially infected whereas EC in other organs are less affected. As in vivo, we found differences in the infection of EC in cell culture. Only brain-derived primary or immortalized EC were found to be permissive to NiV infection. Using a replication-independent fusion assay, we could show that the lack of infection in non-brain EC was due to a lack of receptor expression. The NiV entry receptors ephrinB2 (EB2) or ephrinB3 were only expressed in brain endothelia. The finding that EB2 expression in previously non-permissive aortic EC rendered the cells permissive to infection then demonstrated that EB2 is not only necessary but also sufficient to allow the establishment of a productive NiV infection. This strongly suggests that limitations in receptor expression restrict virus entry in certain EC subsets in vivo, and are thus responsible for the differences in EC tropism observed in human and animal NiV infections.

Figure 1

NiV infection and NiV glycoprotein-mediated cell-to-cell fusion in different model EC. PBMEC, HBMEC, PAEC, MyEnd, Ea.hy 926 and control Vero and HeLa cells were infected with NiV at a MOI of 0.2. (A) At 24 h p.i., cells were fixed, incubated with a NiV-specific guinea pig antiserum and visualized with rhodamine-conjugated secondary antibodies. Nuclei were counterstained with DAPI. (B) At 48 h p.i., viral RNA was isolated from supernatants of infected cells. RT-PCR was performed using NP-specific primers (NPfor binds at bp 1160–1179 and NPrev binds at bp 1271–1292). (C) HeLa cells were cotransfected with plasmids encoding the NiV glycoproteins F and G and were incubated at 33°C. 22 h after transfection, control cells and the different EC types were overlayed with NiV F- and G-expressing HeLa cells. 24 h later, cell-to-cell fusion was visualized by Giemsa staining. Magnification, ×20.

Figure 2

EB2 and EB3 expression in model EC. (A) EC were cultured on coverslips and immunostaining was performed using recombinant EphB4/Fc and rhodamine-conjugated secondary antibodies. Nuclei were visualized by DAPI staining. Magnification, ×100. (B) mRNA was extracted from 5 × 10⁵ cells by standard procedures and subjected to RT-PCR with EB2-specific primers. (C) RT-PCR with EB3-specific primers.

Figure 3

NiV infection of EB2 expressing PAE cells (PAEC-EB2). (A) PAEC and PAEC-EB2 cells were immunostained for EB2 as described in the legend to Fig. 2B. Magnification, ×100. (B) PAEC and PAEC-EB2 were infected with NiV at a MOI of 0.2. At 24 h p.i., cells were fixed and immunostaining was performed as described in the legend to Fig. 1A. Magnification, ×20. (C) 48 h p.i., mRNA was isolated from the cell supernatant, and RT-PCR was performed with NP-specific primers.