Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells

Abstract

The highly pathogenic Nipah virus (NiV) is aerially transmitted and causes a systemic infection after entering the respiratory tract. Airway epithelia are thus important targets in primary infection. Furthermore, virus replication in the mucosal surfaces of the respiratory or urinary tract in later phases of infection is essential for virus shedding and transmission. So far, the mechanisms of NiV replication in epithelial cells are poorly elucidated. In the present study, we provide evidence that bipolar targeting of the two NiV surface glycoproteins G and F is of biological importance for fusion in polarized epithelia. We demonstrate that infection of polarized cells induces focus formation, with both glycoproteins located at lateral membranes of infected cells adjacent to uninfected cells. Supporting the idea of a direct spread of infection via lateral cell-to-cell fusion, we could identify basolateral targeting signals in the cytoplasmic domains of both NiV glycoproteins. Tyrosine 525 in the F protein is part of an endocytosis signal and is also responsible for basolateral sorting. Surprisingly, we identified a dityrosine motif at position 28/29 in the G protein, which mediates polarized targeting. A dileucine motif predicted to function as sorting signal is not involved. Mutation of the targeting signal in one of the NiV glycoproteins prevented the fusion of polarized cells, suggesting that basolateral or bipolar F and G expression facilitates the spread of NiV within epithelial cell monolayers, thereby contributing to efficient virus spread in mucosal surfaces in early and late phases of infection.

FIG. 1.

NiV infection of polarized MDCK cells. MDCK cells were grown on filter supports until full polarization was reached. Then cells either were left uninfected (mock) or were infected with NiV. At 18 h p.i. or 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with a NiV-specific guinea pig antiserum and Alexa Fluor 568-conjugated secondary antibodies. After permeabilization with methanol-acetone, cell junctions were visualized with a monoclonal antibody directed against E-cadherin and with Alexa Fluor 488-conjugated secondary antibodies. Magnification, ×630. Bar, 10 μm.

FIG. 2.

Distribution of F and G proteins in NiV-induced foci. MDCK cells were cultured on filter supports for 7 days and were then infected with NiV. At 18 h p.i., cells were fixed with 4% PFA for 48 h and were then incubated from the apical and basolateral sides with either F-specific (A) or G-specific (B) monoclonal antibodies and Alexa Fluor 568-conjugated secondary antibodies. (Top) Confocal horizontal (xy) sections through the apical part of the cell monolayer. Dashed lines indicate the lines along which vertical sections were recorded. (Bottom) Vertical (xz) sections through the foci. Arrows indicate lateral membranes. Bars, 10 μm.

FIG. 3.

NiV release and syncytium formation at different time points p.i. MDCK cells were cultivated on filter supports until full polarization was reached. Then cells were infected with NiV at a multiplicity of infection of 10. (A) Virus titers in the supernatants were determined by the TCID₅₀ method at 0, 2, 6, 8, 13, 16, 20, 24, 39, and 48 h p.i. (B) To visualize syncytium formation at the indicated time points p.i., cells were fixed, permeabilized with methanol-acetone, and inactivated with 4% PFA. Subsequently, cells were stained with a NiV-specific guinea pig antiserum and Alexa Fluor 568-conjugated secondary antibodies. Magnification, ×200.

FIG. 4.

Surface distribution of wild-type F and G proteins in polarized MDCK cells. (A and B) At 7 days after seeding on filter supports, MDCK cells stably expressing either NiV Fwt (A) or NiV Gwt (B) were incubated with a NiV-specific antiserum from the apical and basolateral sides without prior fixation. Surface-bound antibodies were detected with Alexa Fluor 568-conjugated secondary antibodies. Confocal horizontal sections from top (upper left) to bottom (lower right) are shown. (Insets) Vertical sections. Bars, 20 μm. (C and D) Cell surface proteins were labeled with S-NHS biotin from either the apical (ap) or the basolateral (bas) side. (C) After cell lysis, F and G proteins were immunoprecipitated with NiV-specific antibodies. Precipitates were analyzed by SDS-PAGE under nonreducing (Fwt) or reducing (Gwt) conditions, transferred to nitrocellulose membranes, and probed with peroxidase-conjugated streptavidin. (D) To control surface-selective biotinylation after apical and basolateral labeling, 0.5% of the total-cell lysates was directly subjected to SDS-PAGE, blotting, and streptavidin detection. #, proteins selectively expressed on the apical surface; *, proteins found predominantly after basolateral biotinylation.

FIG. 5.

Surface distribution of 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 mutant NiV glycoproteins. MDCK cells stably expressing either wild-type or mutant NiV F (B) or G (C) proteins were immunostained as described in the legend to Fig. 4. Vertical sections through the cell monolayers are shown. Bar, 10 μm.

FIG. 6.

Fusion activity and surface distribution of wild-type and mutant NiV F and G proteins upon coexpression. (A) Syncytium formation in nonpolarized MDCK cells. Subconfluent MDCK cells stably expressing NiV Fwt were transfected with plasmids encoding either Gwt protein (Fwt + Gwt) or a mutant NiV G protein (Fwt + G_L41/42A or Fwt + G_Y28/29A). At 24 h p.t., syncytia were visualized by staining with a G-specific monoclonal antibody without prior fixation, and the primary antibody was detected with Alexa Fluor 568-conjugated secondary antibodies. Cell nuclei were counterstained with DAPI. Magnification, ×200. (B) Syncytium formation in polarized MDCK cells. MDCK cells stably expressing NiV F protein were grown on filter supports. At 5 days postseeding, cells were transfected with plasmids encoding either wild-type or mutant NiV G. G-positive cells were stained at 48 h p.t. as described above. After permeabilization, cell junctions were visualized with an E-cadherin-specific monoclonal antibody and Alexa Fluor 488-conjugated secondary antibodies. Confocal horizontal sections (top) and side views (bottom) are shown. (C) Distribution of wild-type F and G proteins in polarized MDCK cells upon coexpression. MDCK cells stably expressing Fwt were grown on filter supports until full polarization was reached. Then the cells were transfected with a plasmid encoding Gwt. At 48 h p.t., stably expressed Fwt was surface stained from both sides with F-specific monoclonal antibodies and Alexa Fluor 568-conjugated secondary antibodies. The transiently expressed Gwt was then detected with G-specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. (Left) Confocal xy section through the apical part of the monolayer; (right) vertical section recorded along the line indicated by the blue line in the right panel. (D) Distribution of wild-type G protein upon coexpression with F_Y525A. Polarized Gwt-expressing MDCK cells were transfected with mutant F_Y525A. At 48 h p.t., Gwt was surface stained from both sides with G-specific monoclonal antibodies and Alexa Fluor 568-conjugated secondary antibodies. The transiently expressed F_Y525A was then detected with F-specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. Confocal vertical sections are shown. Bars, 10 μm.