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Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

ABSTRACT: The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding.

SUBMITTER: Mirza AM

PROVIDER: S-EPMC3093860 | biostudies-other | 2011 May

REPOSITORIES: biostudies-other

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Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

Mirza Anne M AM Aguilar Hector C HC Zhu Qiyun Q Mahon Paul J PJ Rota Paul A PA Lee Benhur B Iorio Ronald M RM

The Journal of biological chemistry 20110401 20

The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with e ...[more]

PMID: 21460213

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Project description:The Paramyxoviridae family comprises important pathogens that include measles (MeV), mumps, parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). Paramyxoviral entry into cells requires viral-cell membrane fusion, and formation of paramyxoviral pathognomonic syncytia requires cell-cell membrane fusion. Both events are coordinated by intricate interactions between the tetrameric attachment (G/H/HN) and trimeric fusion (F) glycoproteins. We report that receptor binding induces conformational changes in NiV G that expose its stalk domain, which triggers F through a cascade from prefusion to prehairpin intermediate (PHI) to postfusion conformations, executing membrane fusion. To decipher how the NiV G stalk may trigger F, we introduced cysteines along the G stalk to increase tetrameric strength and restrict stalk mobility. While most point mutants displayed near-wild-type levels of cell surface expression and receptor binding, most yielded increased NiV G oligomeric strength, and showed remarkably strong defects in syncytium formation. Furthermore, most of these mutants displayed stronger F/G interactions and significant defects in their ability to trigger F, indicating that NiV G stalk mobility is key to proper F triggering via moderate G/F interactions. Also remarkably, a mutant capable of triggering F and of fusion pore formation yielded little syncytium formation, implicating G or G/F interactions in a late step occurring post fusion pore formation, such as the extensive fusion pore expansion required for syncytium formation. This study uncovers novel mechanisms by which the G stalk and its oligomerization/mobility affect G/F interactions, the triggering of F, and a late fusion pore expansion step-exciting novel findings for paramyxoviral attachment glycoproteins. IMPORTANCE The important Paramyxoviridae family includes measles, mumps, human parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). The deadly emerging NiV can cause neurologic and respiratory symptoms in humans with a >60% mortality rate. NiV has two surface proteins, the receptor binding protein (G) and fusion (F) glycoproteins. They mediate the required membrane fusion during viral entry into host cells and during syncytium formation, a hallmark of paramyxoviral and NiV infections. We previously discovered that the G stalk domain is important for triggering F (via largely unknown mechanisms) to induce membrane fusion. Here, we uncovered new roles and mechanisms by which the G stalk and its mobility modulate the triggering of F and also unexpectedly affect a very late step in membrane fusion, namely fusion pore expansion. Importantly, these novel findings may extend to other paramyxoviruses, offering new potential targets for therapeutic interventions.

Project description:Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

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Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

Publications

Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

Similar Datasets

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