Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.The zoonotic Hendra virus (HeV) uses the flying fox as a natural host and is highly virulent in humans and horses. Reported cases of HeV transmission in humans have, to date, been sporadic and restricted to Australia (23, 32, 42). HeV was originally discovered in 1994 following the infection and subsequent death of a horse trainer and numerous horses (42, 49). Infection was characterized by the swift onset (7 to 10 days) of acute respiratory disease with clinical symptoms including fever, dizziness, hypotension, and respiratory illness. HeV is closely related to Nipah virus (NiV) which, in its largest outbreak, was responsible for the death of approximately 105 people in Malaysia (46). HeV and NiV (referred to as HNV) collectively form the Henipavirus genus which belongs to the Paramyxoviridae family of single-stranded, negative-sense RNA viruses. Putative new members of this genus have been recently identified in African bats (19).HeV contains attachment (HeV-G) and fusion (HeV-F) membrane glycoproteins which extend from the viral envelope and are required for efficient entry (20). HeV and NiV exhibit a broad tissue tropism which correlates with the use of the widely expressed cell surface glycoproteins ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as functional receptors for HeV-G and NiV-G (referred to as HNV-G). Oligomerization of HeV-G is also critical for productive attachment (1, 4). Despite the shared underlying molecular architecture of the attachment glycoprotein across the paramyxoviruses, there is marked divergence in their cellular receptors. Newcastle disease virus (NDV) and parainfluenza viruses (PIVs) attach to cell surface sialic acid (neuraminic acid), a negatively charged nine-carbon saccharide that is located at the nonreducing termini of glycolipids and N- and O-linked glycans of glycoproteins. In contrast, morbilliviruses such as measles virus (MV), canine distemper virus, and rinderpest virus have been shown to require surface lymphocyte activation molecule (SLAM; CD150) (51, 52) or the complement regulator CD46 for viral attachment (18, 44, 48). Structure-based phylogenetic analysis of available paramyxovirus structures shows that NiV-G and HeV-G are structurally more similar to attachment glycoproteins of sialic acid-binding viruses such as NDV and PIVs than to that of MV (7). We have suggested that the protein-binding capacities of henipaviruses and morbilliviruses have evolved independently, indicating an innate propensity for viruses to acquire novel protein receptor specificity, a characteristic that presents a natural route for the emergence of new pathogenic viruses (7).HeV-G and NiV-G are closely related by sequence (81% identity) and structure (7). This similarity is underscored by the observation that vaccination of cats with recombinant HeV-G can protect against challenge with NiV (39). The similarity is evident in the crystal structures of NiV-G and HeV-G in complex with EFNB2, which both display a common binding mode (7). Knowledge of the molecular determinants of HeV attachment and fusion is of value for the rational development of immunotherapeutic and antiviral reagents with which to target these viruses. Such a structure-based approach to drug design has led to the development of active antivirals against influenza (2). However, in contrast to the rigid carbohydrate binding cleft of influenza neuraminidase, crystallographic analysis of the unbound NiV-G revealed significant plasticity in many of the EFNB2-binding loops which extend from the β-propeller scaffold (10). This information redefines the structural template for rational antiviral drug design against the HNV-G family of viral glycoproteins. Here, we have sought to further refine this template by identifying structural characteristics of HeV attachment.We have crystallized and solved the structure of the globular six-bladed β-propeller domain of HeV-G. Analysis of this structure reveals that, as previously observed for the closely related NiV-G, significant conformational changes occur in the EFNB2 and EFNB3 receptor binding loops between unliganded and receptor-bound structures (10). Additionally, based on the packing of identical HeV-G molecules in the crystal asymmetric unit, we provide a model for the dimeric component of the native oligomeric assembly of the attachment glycoprotein. This model is consistent with oligomeric structures from other paramyxovirus attachment glycoproteins, and we propose that it may represent a component of the biological assembly. Furthermore, we observe that the angle of association between monomeric subunits is much more conserved between glycan-binding hemagglutinin-neuraminidases (HN) than the protein-binding HeV and MV attachment glycoproteins. As a result, we suggest that the acquisition of protein-binding functionality by paramyxoviruses may require structural adaptation, which perturbs the conserved dimeric packing. Together these data support the hypothesis of a globally conserved mode of oligomerization among the paramyxoviruses.