Ephrin-B2 expression critically influences Nipah virus infection independent of its cytoplasmic tail

A correlation of expression levels of cell-surface receptors and infection efficiency has been shown for many different viruses. Increased receptor expression had either a beneficial effect on virus replication or had no effect [36‐43]. To determine the influence of differences in receptor expression on NiV replication, we first analyzed the effect on glycoprotein-mediated fusion in the absence of a NiV infection. To monitor EB2 surface expression, EB2-negative HeLa cells were transfected with increasing amounts of EB2 plasmid DNA (pCAGGS-EB2), and were analyzed at 24 h post transfection (p.t.) by immunostaining (Fig. 1A) and FACS analysis (Fig. 1B). For immunofluorescence staining, living cells were incubated with recombinant EphB4/Fc. Surface-bound EphB4/Fc was then detected with a rhodamine-conjugated anti-human IgG antibody. FACS analysis of surface-expressed EB2 was performed using an EB2-specific antibody and FITC-conjugated anti-goat secondary antibodies. As shown in Fig. 1A and 1B, the number of EB2-positive cells raised with increasing quantities of EB2 DNA. In the sample transfected with 1 μg pCAGGS-EB2, 63.2% of the cells expressed EB2 on the cell surface. The mean fluorescence values, thus the mean receptor densities, were the same in all EB2-transfected cells indicating that cultures with more EB2-positive cells contained an increased total number of cells with higher EB2 expression levels, but maximal EB2 surface expression levels were not upregulated by transfection of more plasmid DNA.

To analyze the ability of cell cultures transfected with different EB2 plasmid concentrations to support NiV glycoprotein-mediated cell-to-cell fusion, constant and optimized ratios of NiV F and G protein encoding plasmids were cotransfected, and syncytia formation was monitored at 24 h p.t. by staining with Giemsa solution (Fig. 1C). Since HeLa cells do not express endogenous EB2, mock-transfected HeLa cells did not support any NiV glycoprotein-specific fusion (Fig. 1C, mock). As expected, EB2 transfection was required to render cells susceptible for cell-to-cell fusion. However, pronounced syncytia formation was only seen in cells transfected with low amounts of pCAGGS-EB2, higher amounts interfered with efficient cell-to-cell fusion. When we determined the mean size of syncytia by counting and averaging the number of nuclei per syncytium (Fig. 1D), we found the largest syncytia (14 nuclei) in HeLa cells transfected with only 0.05 μg pCAGGS-EB2. Then, the size decreased stepwise with increasing DNA amounts down to 4.9 nuclei per syncytium in cells transfected with 1 μg pCAGGS-EB2.

To test if differences in EB2 expression also affect syncytium induction in NiV permissive cells expressing endogenous EB2, cotransfection of the NiV glycoprotein genes in addition to various amounts of pCAGGS-EB2 was performed in Vero cells. As anticipated, NiV F and G induced syncytium formation in mock-transfected cells (Fig. 2A, mock). Supplemental expression of exogenous EB2, even at the lowest concentration tested (0.1 μg DNA), resulted in decreased cell-to-cell fusion (Fig. 2A and 2B). In accordance with HeLa cells, EB2 overexpression in Vero cells clearly led to downregulation of NiV glycoprotein-induced syncytia formation. FACS analysis to quantify EB2 expression levels in transfected Vero cells again showed that with rising amounts of EB2 DNA an increasing percentage of Vero cells (up to 40% in cells transfected with 1 μg DNA) expressed EB2 at higher surface densities (data not shown). The mean fluorescence values in this cell population expressing additional plasmid-encoded EB2 was about 10-fold higher than in Vero cells expressing endogenous EB2 only (mock transfected cells). To exclude that these higher EB2 expression levels have a general downregulating effect on paramyxovirus cell-to-cell fusion, we investigated the effect of EB2 overexpression on syncytium formation caused by the measles virus Edmonston (MV_Edm) glycoproteins F and H. MV_Edm does not bind to EB2 but uses CD46 as entry receptor which is also endogenously expressed in Vero cells [44]. Cells were transfected with the MV_Edm F and H genes together with different amounts of pCAGGS-EB2 and syncytia formation was analyzed at 15 h p.t.. In contrast to what we had observed for NiV, EB2 overexpression had no negative effect on MV_Edm glycoprotein-mediated cell-to-cell fusion (Fig. 2C). This demonstrates that higher amounts of EB2 do not generally interfere with paramyxoviral glycoprotein-induced fusion but specifically inhibit NiV F- and G-mediated syncytia formation. When we expressed the NiV F and G protein in the presence of increasing amounts of CD46 we again did not see any effect on syncytia formation (Fig. 2D). Therefore, the downregulating effect on NiV glycoprotein-mediated fusion by EB2 is specific.

To determine if EB2 overexpression affects surface expression of the NiV F or G protein, Vero cells were cotransfected with constant amounts of plasmids encoding the NiV glycoproteins F and G in addition to varying amounts of pCAGGS-EB2. At 24 h p.t., cells were surface biotinylated followed by immunoprecipitation of F and G proteins. After separation by SDS-PAGE and blotting to nitrocellulose, surface expressed NiV glycoproteins were detected by IRDye 800-conjugated streptavidin. As shown in Fig. 3A and 3B, EB2 overexpression reduced the expression levels of F and G protein on the cell surface in a concentration-dependent manner. The finding that surface expression of the MV glycoproteins F and H was not influenced by EB2 transfection (Fig. 3C) clearly suggests that surface downregulation of NiV glycoprotein complexes is due to specific interactions with EB2.

To analyze the effects of additional EB2 expression in the context of a NiV infection, Vero cells were transfected with different amounts of the EB2-encoding plasmid, transferred to the BSL4 facility and subsequently infected with NiV at a multiplicity of infection (MOI) of 1. NiV-positive cells were stained by indirect immunofluorescence analysis at 24 h post infection (p.i.) to reveal the size and the number of syncytia. To quantify virus production, virus titers in the supernatant were determined by the TCID₅₀ method. For immunostaining, NiV-infected cells were fixed and incubated with a NiV-specific guinea pig antiserum and rhodamine-conjugated secondary antibodies. Cell nuclei were counterstained with DAPI. Merged pictures of the rhodamine and DAPI fluorescence channels are shown in Fig. 4A and demonstrate that all Vero cell cultures transfected with different amounts of pCAGGS-EB2 could be infected with NiV. However, the number and size of syncytia appeared to be reduced in cells transfected with pCAGGS-EB2. This was confirmed by determining the average size of the NiV-induced syncytia (Fig. 4B). Whereas syncytia in mock-transfected cells contained about 50 nuclei in average, cells transfected with 0.25 μg, 0.5 μg or 1 μg of EB2 plasmid DNA produced syncytia with 22, 19 or 15 nuclei, respectively. To evaluate if differences in EB2 expression also affect virus entry, the total number of syncytia in each sample was counted and was also found to be reduced in EB2-transfected cells (Fig. 4C). Since one syncytium originates from one initially infected cell, this finding clearly indicates that not only NiV-mediated cell-to-cell fusion but also virus entry is impaired in Vero cells overexpressing EB2. In agreement with the decreased total number of infected cells and the less efficient spread via cell-to-cell fusion, the amount of infectious NiV particles released into the cell supernatant was also drastically diminished. Virus titers were reduced by more than 100-fold (Fig. 4D). To examine if inhibition of infection by EB2 overexpression is specific for NiV, control studies were performed with MV_Edm. Vero cells transfected with various quantities of pCAGGS-EB2 were infected with MV_Edm at a MOI of 1. Since no infectious virus could be detected at 24 h p.i., the amount of virus particles released from the cells was determined at 42 h p.i. by plaque assay. In contrast to NiV, MV_Edm virus titers in the supernatant of mock- and EB2-transfected Vero cells were similar (Fig. 4E) demonstrating that MV_Edm infection was not affected by variations in EB2 expression. We also analyzed MV_Edm replication in the presence of increased levels of its own receptor CD46, but we did not observe any negative influence of CD46 overexpression on productive MV_Edm infection (data not shown). We thus conclude that the negative effect of EB2 overexpression on productive virus replication is specific for NiV.

Clustering of EB2 by NiV G protein binding during virus entry may be an essential component of these processes and might trigger EB2 signaling. Supporting this idea, it was recently shown that the critical residues in EB2 involved in interaction with NiV G are the same required for interaction with the EphB2 receptor [27]. After NiV G-induced EB2 clustering, NiV entry might be influenced by proteins interacting with the catalytic domain of the EB2 cytoplasmic tail, such as proteins containing PDZ domains which can stabilize high-ordered clustering into oligomeric arrays [45]. The density of this clustering or effects of EB2 signaling on actin cytoskeleton rearrangements may modulate the efficiency of virus-cell fusion. Therefore, the negative effect of EB2 overexpression on NiV entry could be the result of an overshooting EB2 signaling. To evaluate this idea, we decided to study the influence of a tail-truncated ΔcEB2 that lacks the C-terminal 67 amino acids on NiV replication [46] (Fig. 5A). First, we analyzed the effect of increased ΔcEB2 surface expression on NiV glycoprotein-mediated cell-to-cell fusion in Vero cells by transfecting the NiV F and G genes in addition to different amounts of EB2 or ΔcEB2 plasmid DNA. As with wildtype EB2, an expression level-dependent reduction of the size of syncytia was observed in ΔcEB2-expressing cells (Fig. 5B and 5C). To investigate the effect of increased amounts of tail-truncated EB2 on NiV infection, ΔcEB2-transfected Vero cells were infected with NiV and syncytium formation and virus production was monitored at different time points p.i.. The quantitative analysis at 15 h p.i. is depicted in Fig. 6 and shows that overexpression of tail-truncated EB2 affected productive NiV infection to similar extents as full-length EB2 (Fig. 4). Transfection of only 0.25 μg of ΔcEB2 plasmid DNA already drastically interfered with NiV-induced cell-to-cell spread (Fig. 6B) and reduced the release of infectious viruses by 2 to 3 log steps (Fig. 6C). Higher DNA concentrations had no further substantial effects. This indicates that receptor overexpression can downregulate productive NiV infection by interfering with virus entry and F- and G-mediated cell-to-cell fusion but cannot completely prevent infection.

The finding that overexpression of ΔcEB2 interfered with NiV infection suggests that tail-truncated EB2 can also function as NiV binding partner. To directly test if ΔcEB2 can be used as NiV entry receptor or if it even provides a more effective receptor than full-length EB2 because it no longer functions in signaling, we analyzed NiV infection in HeLa cells and porcine aortic endothelial cells (PAEC) stably expressing either wildtype or a tail-truncated ΔcEB2. As we got similar results for both cell lines, only the results obtained for the PAEC, a well-studied cell line in terms of EB2 functions [46] are shown. To control the protein expression, EB2 and ΔcEB2 proteins were immunoprecipitated from cell lysates and subjected to western blot analysis using an EB2-specific antibody. As shown in Fig. 7A, expression of EB2 and ΔcEB2 in stably transfected PAEC is comparable. To characterize surface localization of EB2 and the tail-truncated variant, double-labeling experiments for EB2 and VE-cadherin, a marker protein for adherens junctions in endothelial cells, were performed. PAEC-EB2 and -ΔcEB2 were seeded on porous filter membranes and cultured for 7 days to form a polarized endothelial cell monolayer. Surface-expressed EB2 and ΔcEB2 was detected by incubation with EphB4/Fc on ice and subsequent treatment with rhodamine-conjugated anti-human IgG antibodies. Then, cells were permeabilized and stained with a VE-cadherin antibody and a FITC-conjugated secondary antibody. Analysis of vertical sections through the endothelial cell monolayers identified an equal luminal expression of both EB2 and ΔcEB2 (Fig. 7B). To control the loss of function of the tail-truncated EB2, an EphB4 receptor body uptake experiment was performed [33]. For this, PAEC-EB2 and PAEC-ΔcEB2 were incubated with recombinant EphB4/Fc for 1 h at 37°C to allow binding of EphB4/Fc and co-endocytosis of EB2 and EphB4/Fc to occur. Surface-remained EphB4/Fc was visualized by incubation with rhodamine-conjugated secondary antibodies at 4°C. After fixation and permeabilization, intracellular EphB4/Fc was stained with FITC-conjugated secondary antibodies. In PAEC stably expressing wildtype EB2, numerous fluorescent intracellular vesicles (green dots) were found (Fig. 7C, PAEC-EB2). In contrast, in PAEC-ΔcEB2 expressing cells, no intracellular complexes were detected demonstrating that tail-truncated EB2 is no longer endocytosed. To analyze the receptor function of tail-truncated EB2, PAEC-EB2 or PAEC-ΔcEB2 were infected with NiV at a MOI of 1. To monitor the infection at 24 h p.i., NiV-positive cells and syncytia were detected by immunostaining and virus titers in the supernatant were quantified by the TCID₅₀ method. As shown in Fig. 7D, extensive cell-to-cell fusion could be detected in both PAEC-EB2 and PAEC- ΔcEB2. The average size as well as the number of syncytia was similar. Accordingly, the amount of infectious virus particles released into the supernatant was the same (3.1 × 10⁶ TCID₅₀/ml for both cell lines). The finding that there are no substantial differences in the amount of initially infected cells, NiV-mediated cell-to-cell fusion and virus titers demonstrates that the tail-truncated ΔcEB2 can fully function as NiV entry receptor in endothelial cells, the predominant target cells of the NiV infection in vivo.

Vero (African green monkey kidney) and HeLa (human cervical cancer) cells were maintained in Dulbecco's modified minimal essential medium (DMEM, Gibco) containing 10% fetal calf serum (FCS), 4 mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. PAE (porcine aortic endothelial) cells were cultured in DMEM/F12 + GLUTAMAX (Gibco) supplemented with 10% FCS, penicillin and streptomycin.

The NiV strain used in this study was isolated from human brain tissue (kindly provided by J. Cardosa) and propagated as described earlier [73]. For NiV infection, confluent cell monolayers of different cell lines were infected at a multiplicity of infection (MOI) of 1. After incubation for 1 h at 37°C, inocula were removed, cells were washed twice and then cultured with medium containing 2% FCS at 37°C. All work with live NiV was performed under BSL4 conditions.

Measles virus vaccine strain Edmonston (MV_Edm) was grown and propagated on Vero cells as described previously [74]. For MV_Edm infection studies, cells were infected with MV_Edm at a MOI of 1. After incubation for 2 h at 37°C, virus was removed by extensive washings and cells were incubated with medium containing 2% FCS at 33°C.

The NiV F and G glycoprotein open reading frames (GenBank™ accession number AF212302) were subcloned into the pczCFG5 expression vector as described by Moll et al. [75]. Cloning of the MV_Edm glycoprotein genes (F and H) into the pCG expression vector has been described earlier [76]. The human EB2 gene and a C-terminally truncated EB2 version (encoding amino acid residues 1–266), designated as ΔcEB2 [46], were subcloned into the NotI site and SacI site of a pCAGGS expression vector [77]. Human CD46 gene was cloned into the pHßAPr.1-neo expression vector as specified previously [78].

Transfections of Vero and HeLa cells were performed by using the cationic liposome based transfection reagent Lipofectamine 2000 (Invitrogen) according to the supplier's instructions. Stably EB2- and ΔcEB2-expressing PAE cells were constructed as described by Füller et al. [46].

HeLa cells transiently expressing varying amounts of EB2 were cultured on coverslips. At 24 h post transfection (p.t.), recombinant mouse EphB4/Fc, a soluble EB2 receptor fused to the Fc region of human IgG (R&D Systems) was added at a concentration of 2 μg/ml for 1 h at 4°C. Bound EphB4/Fc was stained with rhodamine-conjugated anti-human IgG antibodies on ice (dilution 1:200; Jackson ImmunoResearch). Nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Representative merged images of the DAPI and rhodamine channels were recorded with a Zeiss Axiovert 200 M microscope.

Stably EB2- and ΔcEB2-expressing PAE cells were cultured on 0,4 μm-pore size ThinCert polyethylenterephthalat filter supports (Greiner Bio-one). After 7 days, the apical and basolateral surfaces were incubated with recombinant EphB4/Fc (2 μg/ml; 2 h at 4°C) and rhodamine-conjugated secondary antibodies (dilution 1:200; 2 h at 4°C). To visualize the adherens junctions, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100 for 15 min and subsequently treated with anti VE-cadherin antibodies (Santa Cruz) at a dilution of 1:100 for 2 h at 4°C and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibodies (dilution 1:200; 2 h at 4°C; Jackson ImmunoResearch). Filter membranes were analyzed with a confocal laser scanning microscope (LSM 510, Zeiss).

HeLa or Vero cells were transfected with different amounts of EB2 DNA. At 24 h p.t., cells were detached with phosphate buffered saline (PBS) containing 5 mM EDTA, washed twice and 5 × 10⁵ cells were subsequently incubated with an EB2-specific antibody (dilution 1:10; R&D Systems) for 45 min at 4°C. Primary antibodies were detected by FITC-conjugated anti-goat IgG secondary antibodies (dilution 1:100; Jackson ImmunoResearch) and flow-cytometric analyses were carried out with a FACScan (Guava Technologies). Since it has been shown that coexpression of NiV G protein did not influence the expression of EB2 on the cell surface [66], FACS analysis was performed in cells expressing EB2 only.

As it has been reported for the closely related HeV glycoproteins that the ratio of F to G plasmids transfected into cells can influence the efficiency of membrane fusion [79], we first tested the capacity of varying levels of pczCFG5-F and pczCFG5-G transfected into Vero cells to mediate cell-to-cell fusion. In agreement with what has been shown for the HeV glycoproteins, increased fusion was observed with greater amounts of the G protein plasmid. Since NiV glycoprotein-mediated syncytia formation was found to be maximal at a 1:5 ratio of NiV F to NiV G plasmid DNA, cells were cotransfected with constant amounts of plasmids encoding either the NiV glycoproteins F and G at this ratio or the MV glycoproteins F and H (optimal ratio of 1:1), in addition to varying amounts of pCAGGS-EB2, pCAGGS-ΔcEB2 or pHßAPr.1-neo-CD46, respectively. At 24 h (NiV) or 15 h (MV) p.t., cells were fixed with ethanol for 10 min and incubated with a 1:10 diluted Giemsa staining solution for 30 min to visualize syncytium formation. Representative microscopic fields were photographed. To quantify the size of syncytia, the number of nuclei per syncytium of twenty randomly chosen syncytia were counted and averaged.

Cell surface proteins were labeled with sulfosuccinimidobiotin (S-NHS-biotin; Pierce) and subsequently lysed in radioimmunoprecipitation assay buffer (RIPA) as described previously [17]. Immunoprecipitation of NiV F from surface-biotinylated cells was carried out with an F-specific antibody directed against amino acids 523 to 541 of the NiV F cytoplasmic domain (dilution 1:100; ImmunoGlobe Antikörpertechnik), and NiV G was immunoprecipitated with a polyclonal NiV-specific antiserum (dilution 1:350). MV glycoproteins were isolated using F- or H-specific monoclonal antibodies [76]. Immunoprecipitates were separated on a 12% polyacrylamide gel under reducing (NiV G, MV F and H) or non-reducing conditions (NiV F) and blotted to nitrocellulose. Nonspecific binding sites were blocked with 5% nonfat dry milk in PBS. To detect surface-biotinylated NiV and MV glycoproteins, blots were incubated with IRDye 800-conjugated streptavidin for 45 min at 4°C (Rockland; dilution 1:8000). Fluorescent signals were analyzed using a LiCor-Odyssey infrared imaging system (LI-COR Biosciences GmbH).

Vero cells grown on coverslips were transfected with varying amounts of pCAGGS-EB2 or -ΔcEB2. At 15 h p.t., infection with NiV was performed as described above. NiV-positive cells or syncytia were detected by indirect immunofluorescence as described recently [15]. Briefly, after fixation with 4% PFA for 48 h, cells were permeabilized with methanol-acetone and incubated with a NiV-specific guinea pig antiserum (dilution 1:1000) for 1 h at 4°C. Primary antibodies were detected with rhodamine-conjugated anti-guinea pig IgG antibodies (Jackson ImmunoResearch; dilution 1:200; 45 min at 4°C). Nuclei were counterstained by DAPI. Images were recorded using a Zeiss Axiovert 200 M microscope.

The size of NiV-induced syncytia was quantified as described above. The number of initially infected cells in each sample was determined by counting the number of NiV-positive syncytia. To quantify virus release, virus titers in the supernatant were calculated by the 50% tissue culture infective dose (TCID₅₀) method on Vero cells [80].

Stably EB2- and ΔcEB2-expressing PAEC were lysed in immunoprecipitation buffer (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7,4). EB2- and ΔcEB2 were immunoprecipitated with EphB4/Fc (dilution 1:100) and 100 μl of a suspension of protein A/G sepharose CL-4B (Pierce). After three washes, immunocomplexes were suspended in reducing sample buffer. Precipitates were subsequently separated on a 10% polyacrylamide gel and transferred to nitrocellulose. The blot was probed with 0,3 μg anti-EB2 (R&D Systems) followed by incubation with peroxidase-conjugated anti-goat IgG antibodies (dilution 1:4000; Jackson ImmunoResearch). EB2 and ΔcEB2 proteins were visualized with the enhanced chemoluminescence system (SuperSignal^® West Pico Chemoluminescent Substrate; Pierce) by exposure to an autoradiography film (GE Healthcare).

Stably EB2- and ΔcEB2-expressing PAEC were seeded on coverslips and grown to subconfluency. Cells were then incubated with 2 μg/ml recombinant EphB4/Fc for 1 h at 37°C to allow binding and endocytosis to proceed. Surface-remained EphB4/Fc was stained with rhodamine-conjugated anti-human IgG antibodies (dilution 1:50) for 90 min at 4°C. After fixation and permeabilization with methanol-acetone, internalized EphB4/Fc was detected by FITC-conjugated anti-human IgG antibodies (dilution 1:500, Jackson ImmunoResearch) for 35 min at 4°C. Images of representative cells were recorded using a Zeiss Axiovert 200 M microscope.