Functional assessment of the glycoproteins of a novel Hendra virus variant reveals contrasting fusogenic capacities of the receptor-binding and fusion glycoproteins

Abstract

A novel Hendra virus (HeV) genotype (HeV genotype 2 [HeV-g2]) was recently isolated from a deceased horse, revealing high-sequence conservation and antigenic similarities with the prototypic strain, HeV-g1. As the receptor-binding (G) and fusion (F) glycoproteins of HeV are essential for mediating viral entry, functional characterization of emerging HeV genotypic variants is key to understanding viral entry mechanisms and broader virus-host co-evolution. We first confirmed that HeV-g2 and HeV-g1 glycoproteins share a close phylogenetic relationship, underscoring HeV-g2's relevance to global health. Our in vitro data showed that HeV-g2 glycoproteins induced cell-cell fusion in human cells, shared receptor tropism with HeV-g1, and cross-reacted with antibodies raised against HeV-g1. Despite these similarities, HeV-g2 glycoproteins yielded reduced syncytia formation compared to HeV-g1. By expressing heterotypic combinations of HeV-g2, HeV-g1, and Nipah virus (NiV) glycoproteins, we found that while HeV-g2 G had strong fusion-promoting abilities, HeV-g2 F consistently displayed hypofusogenic properties. These fusion phenotypes were more closely associated with those observed in the related NiV. Further investigation using HeV-g1 and HeV-g2 glycoprotein chimeras revealed that multiple domains may play roles in modulating these fusion phenotypes. Altogether, our findings may establish intrinsic fusogenic capacities of viral glycoproteins as a potential driver behind the emergence of new henipaviral variants.

Importance: HeV is a zoonotic pathogen that causes severe disease across various mammalian hosts, including horses and humans. The identification of unrecognized HeV variants, such as HeV-g2, highlights the need to investigate mechanisms that may drive their evolution, transmission, and pathogenicity. Our study reveals that HeV-g2 and HeV-g1 glycoproteins are highly conserved in identity, function, and receptor tropism, yet they differ in their abilities to induce the formation of multinucleated cells (syncytia), which is a potential marker of viral pathogenesis. By using heterotypic combinations of HeV-g2 with either HeV-g1 or NiV glycoproteins, as well as chimeric HeV-g1/HeV-g2 glycoproteins, we demonstrate that the differences in syncytial formation can be attributed to the intrinsic fusogenic capacities of each glycoprotein. Our data indicate that HeV-g2 glycoproteins have fusion phenotypes closely related to those of NiV and that fusion promotion may be a crucial factor driving the emergence of new henipaviral variants.

Keywords: Hendra virus; Henipavirus; fusion protein; paramyxovirus; receptor; receptor-binding protein; syncytia.

Fig 1

Protein sequence alignments between HeV-g2 and other HNV glycoproteins. (A) Percent identities among glycoprotein amino acid sequences of two NiV strains (Malaysian and Bangladesh isolates), two HeV strains, and two other HNVs, CedV and MojV. (B and C) Phylogenetic trees comparing glycoprotein amino acid sequences of representative strains from NiV and HeV outbreaks. CedV glycoproteins serve as the outgroup. Branch support values are written in red font and located next to each node. (D and E) Schematics of HeV-g2 G and F proteins. The small blue vertical lines indicate mutated residues as compared to HeV-g1. Sticks with filled circles indicate potential N-X-S/T N-glycosylation sites, and sticks with open circles indicate a loss of potential N-X-S/T N-glycosylation sites as compared to HeV-g1.

Fig 2

Oligomeric states, receptor tropism, and antigenic profiles of HeV-g1 and HeV-g2 G and F. (A) Representative Western blot analysis of HeV-g1 G and HeV-g2 G in non-reducing and reducing conditions. G tetramers (G_Tet), dimers (G_Dim), and monomers (G_Mon) were observed in the non-reducing gel blot. (B) Representative Western blot of HeV-g1 and HeV-g2 F proteins in a reducing gel with F₀ and F₂ bands indicated. (C) Densitometric analysis of G_Tet, G_Dim, and G_Mon bands (from panel A) as percentages of total G = G_Tet + _GDim + G_Mon. (D) Analysis of F₂ bands (from panel B) as percentages of total F = F₀ + F₂. (E) Relative levels of G cell surface expression (G_CSE) measured via flow cytometry as the mean fluorescence intensity (MFI) was normalized to HeV-g1 G_CSE. Relative levels of ephrinB2 or B3 binding were first normalized to the levels of G_CSE of the respective HeV and then normalized to the mean HeV-g1 ephrin binding. (F) Relative levels of G_CSE were first normalized to the mean CedV G_CSE, set to 100%. Relative levels of ephrinB1 binding were first normalized to the G_CSE of the respective HNV and then normalized to the mean CedV ephrinB1 binding. (G) Polyclonal anti-HeV-g1 sera taken from two different rabbits (Rb 836 and 837) immunized with HeV-g1 F and G DNA expression plasmids cross-react with HeV-g2 F and G. Relative G_CSE and F_CSE were normalized to mean HeV-g1 G_CSE and mean F_CSE levels (set to 100%), respectively. Relative levels of serum binding were first normalized to CSE of the respective protein and then normalized to mean HeV-g1 serum binding. Statistical significance was determined via one-way analysis of variance with Tukey’s multiple comparison test. A P value of >0.05 is indicated as ns, and other P values are given the following indicators: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, non-significant.

Fig 3

Cell-cell fusion phenotypes from homotypic and heterotypic combinations of HeV-g1 and HeV-g2 glycoproteins. (A) Relative levels of G cell surface expression (G_CSE), F_CSE, and syncytia counts normalized to their respective HeV-g1 values. (B) Fusion indices (FI_G and FI_F) normalized to the mean HeV-g1 FI levels set to 100%. Fusion indices were calculated by dividing syncytia counts by the respective G_CSE (FI_G) or F_CSE (FI_F) levels and then normalizing the fusion indices to the mean HeV-g1 fusion indices set to 100%. (C) Representative syncytia images in HEK 293T cells. Statistical significance was determined via one-way analysis of variance with Tukey’s multiple comparison test. Only significant P values are shown with the following indicators: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig 4

Cell-cell fusion phenotypes using heterotypic combinations of HeV-g1, HeV-g2, and NiV glycoproteins. (A) Relative fusion indices normalized to the mean HeV-g1 FIs (calculated as described in Fig. 3). (B) Representative syncytia images in HEK 293T cells. (C) Schematic of relative intrinsic fusogenicities of HNV glycoproteins, where increasing bar width corresponds to increased fusion levels. Statistical significance was determined via two-way analysis of variance with Tukey’s multiple comparison test. Only significant P values are shown with the following indicators: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig 5

Cell-cell fusion activities of HeV glycoprotein chimeras. (A and B) Schematics of the chimeric HeV glycoproteins. Chimeric HeV G glycoproteins were constructed by swapping the head domains of HeV-g1 G and HeV-g2 G, yielding the HeV-g1-167-g2 G and HeV-g2-167-g1 G chimeras. Chimeric HeV F glycoproteins were constructed by swapping the transmembrane (TM) and cytoplasmic tail (CT) domains of HeV-g1 F and HeV-g2 F, yielding the HeV-g1-488-g2 F and HeV-g2-488-g1 F chimeras. (C) Relative FI_G normalized to HeV-g1 FI_G and relative FI_F normalized to HeV-g1 FI_F across HeV G chimeras. (D) Relative FI_G normalized to HeV-g1 FI_G and relative FI_F normalized to HeV-g1 FI_F across HeV F chimeras. Statistical significance was determined via two-way analysis of variance with Tukey’s multiple comparison test. Only significant P values are shown with the following indicators: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.