Mutations in the G-H loop region of ephrin-B2 can enhance Nipah virus binding and infection

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses classified in the genus Henipavirus of the family Paramyxoviridae. The entry of henipaviruses occurs through a pH-independent membrane-fusion mechanism mediated by the cooperation of the viral attachment (G) and fusion (F) envelope glycoproteins following virion binding to susceptible host cells. Virus attachment is mediated by the interaction of the G glycoprotein with ephrin-B2 or ephrin-B3, which were identified as the functional receptors of henipavirus. Several residues of the G glycoprotein that are important for receptor binding have been determined through mutagenesis and structural analyses; however, similar approaches have not been carried out for the viral receptor ephrin-B2. Here, an alanine-scanning mutagenesis analysis was performed to identify residues of ephrin-B2 which are critical for NiV binding and entry by using an NiV-F- and -G-glycoprotein pseudotyped lentivirus assay. Results indicated that the G-H loop of ephrin-B2 was indeed critical for the interaction between ephrin-B2 and NiV-G. Unexpectedly, however, some alanine-substitution mutants located in the G-H loop enhanced the infectivity of the NiV pseudotypes, in particular an L124A mutation enhanced entry >30-fold. Further analysis of the L124A ephrin-B2 mutant demonstrated that an increased binding affinity of the mutant receptor with NiV-G was responsible for the enhanced infectivity of both pseudovirus and infectious virus. In addition, cell lines that were stably expressing the L124A mutant receptor were able to support NiV infection more efficiently than the wild-type molecule, potentially providing a new target-cell platform for viral isolation or virus-entry inhibitor screening and discovery.

Fig. 1.

NiV glycoprotein pseudotyped virus infection of HeLa-USU cells expressing wild-type (wt) or mutant ephrin-B2. NiV-G and NiV-F glycoproteins were pseudotyped onto a human immunodeficiency virus (HIV)-1 based core virus using the NL4-3-Luc-E⁻R⁺ backbone (NiVpp) and used to infect HeLa-USU cells transfected with either wt or mutant ephrin-B2. Entry efficiency of NiVpp was measured by luciferase reporter-gene assay and is expressed as a percentage of wt ephrin-B2 (100 %). The enhanced infectivity of cells expressing some of the mutant ephrin-B2 molecules is highlighted by using a split y-axis. Data shown are the mean of triplicates±sd and one representative experiment of three is shown. (a) Schematic of ephrin-B2. Secondary structure elements are shown with an arrow (β-sheet) and black box (α-helix). The asterisks indicate residues that were targeted by alanine-scanning mutagenesis. (b) Mutations located within the G–H loop of ephrin-B2. (c) Mutations located outside the G–H loop of ephrin-B2.

Fig. 2.

Western blot analysis of ephrin-B2 expression. (a) Equal amounts of a plasmid encoding either the wt or a mutant ephrin-B2 gene were transfected into equivalent cultures of HeLa-USU cells. Following expression for 48 h, cells were lysed and equal amounts (volume in µl) of the various lysates were resolved by using SDS-PAGE and transferred onto a PVDF membrane. Duplicate membranes were probed with goat anti-mouse ephrin-B2 (top) or mouse anti-human β-actin (bottom), respectively. (b) Quantification of the expression of the wt and mutant ephrin-B2. The bands in (a) were quantified by densitometry using the ImageJ program (version 1.44) by using values obtained from densitometric measurements of the bands. The values of the ephrin-B2 mutations are expressed as a percentage of wt ephrin-B2 (100 %). The data were normalized with the corresponding values for β-actin.

Fig. 3.

Immunofluorescence detection and flow cytometry of ephrin-B2 expression on cell surface. HeLa-USU cells were transfected with the indicated expression plasmids. Following expression for 24 h, cells were incubated with goat anti-ephrin-B2 antibody, followed by probing with FITC-conjugated donkey anti-goat IgG. The surface expression of wt or mutant ephrin-B2 was detected by confocal immunofluorescence microscopy (a) and quantified by flow cytometry (b). (a) The expression plasmids are shown at the left side of the panels. The columns (from left to right) represent staining of expressed ephrin-B2 (FITC, in green), cell nuclei (Hoechst 33258, in blue) and the superimposed images of the two. (b) Surface expression of wt or mutant ephrin-B2 was quantified by flow cytometry. The data are shown as the mean fluorescence intensity of the transfected cells indicated.

Fig. 4.

Kinetic analysis of NiV-glycoprotein pseudovirus entry into HeLa-USU cells expressing wt or mutant L124A ephrin-B2. (a) Various pseudovirus preparations were adsorbed to ephrin-B2-expressing cells at 4, 25 or 37 °C for 1 h. The inoculum was removed and cells were washed with PBS and cultured with fresh media at 37 °C. Entry efficiency of the NiVpp at the different incubation temperatures was measured by luciferase reporter-gene assay at 48 h post-infection (p.i.) and is expressed as the percentage of wt ephrin-B2 at the corresponding temperature (100 %). (b) Pseudotyped viruses were adsorbed to ephrin-B2-expressing cells at 37 °C for different time intervals, and entry efficiency of the NiVpp was measured by luciferase reporter-gene assay at 48 h p.i. and is expressed as the percentage of wt ephrin-B2 at the corresponding time point (100 %). Error bars indicate sd.

Fig. 5.

Binding kinetic analysis of NiV-G to wt or mutant L124A ephrin-B2 by bio-layer interferometry. (a) Real-time binding analysis of NiV-G to the wt or mutant L124A ephrin-B2. Bio-layer interferometry was used to measure the binding kinetics of soluble NiV-G to both ephrin-B2 (wt) and its mutant (L124A) in response units (nm). Biotinylated soluble NiV glycoprotein G (sNiV-G) was immobilized to streptavidin-coated biosensors, and the binding of wt and mutant ephrin-B2 was assessed at the indicated concentrations. (b) Steady-state analysis of real-time binding data obtained with the Octet Red system. The plots in each panel show response versus protein concentration curves derived from the raw binding data. One representative experiment of two is shown.

Fig. 6.

NiV-glycoprotein-mediated cell-to-cell fusion in HeLa-USU cells transfected with wt or L124A mutant ephrin-B2. (a) HeLa-USU cells were co-transfected with plasmids encoding NiV-F, NiV-G and the indicated ephrin-B2 genes. Cells were fixed 24h post-transfection and nuclei were visualized by Hoechst 33258 staining. Twenty randomly selected syncytia were photographed. (b) Syncytium formation in HeLa-USU cells expressing wt or mutant ephrin-B2 was measured by counting the mean number of nuclei per syncytium in 20 randomly selected syncytia. Data shown are the means of 20 syncytia±sem. Statistical analysis was performed by using Student’s t-tests with spss (version 13.0). Results where P<0.05 were considered to be significant.

Fig. 7.

Infection by NiV or HeV in HeLa-USU cells stably expressing wt (USU-hB2) or L124A mutant (USU-hB2 m) ephrin-B2. Cells were seeded at 20 000 cells per well 1 day before infection. Cells were infected with 500 TCID₅₀ of NiV (a) or HeV (b) in 50 µl. Virus and cells were incubated for the time indicated on the x-axis and then virus was washed off by washing five times with PBS. DMEM+10 % FCS was added to wells and the cells were incubated for 24 h. Cells were then fixed and infected cells visualized by immunofluorescence by using polyclonal rabbit anti-HeV P antisera. Infected cell foci were counted microscopically (n = 4). Statistical analysis was performed by using Student’s t-tests with spss.