Role of the Two Sialic Acid Binding Sites on the Newcastle Disease Virus HN Protein in Triggering the Interaction with the F Protein Required for the Promotion of Fusion

Abstract

Newcastle disease virus (NDV)-induced membrane fusion requires an interaction between the hemagglutinin-neuraminidase (HN) attachment and the fusion (F) proteins, triggered by HNs binding to receptors.NDV HN has two sialic acid binding sites: site I, which also mediates neuraminidase activity, and site II, which straddles the membrane-distal end of the dimer interface. By characterizing the effect on receptor binding avidity and F-interactive capability of HN dimer interface mutations, we present evidence consistent with (i) receptor engagement by site I triggering the interaction with F and (ii) site II functioning to maintain high-avidity receptor binding during the fusion process.Newcastle disease virus (NDV) is a member of the Paramyxoviridae family of negative-strand RNA viruses (12). These viruses enter and spread between cells by virus-cell and cell-cell fusion, respectively, triggered by receptor binding. However, receptor binding and fusion are mediated by two different glycoproteins, the attachment and fusion (F) proteins, respectively, requiring a mechanism by which the two processes can be linked. This is accomplished by a virus-specific interaction between the two proteins (9,10,13,19).The ectodomain of paramyxovirus attachment proteins, including the NDV hemagglutinin-neuraminidase (HN), consists of a long stalk connected to a terminal globular domain (12). Whereas the HN globular domain mediates binding to sialic acid receptors, the stalk mediates the interaction with the homologous F protein (5,15,16). However, the mechanism by which receptor binding triggers fusion is not well understood. The globular head consists of a six-bladed ␤-sheet propeller with a centrally located sialic acid binding site (site I) that also mediates neuraminidase (NA) activity (4). Many mutations in this site, including K236R, abolish receptor binding, NA, and fusion (7), as well as the ability of HN to interact with F at the cell surface (14). These findings, along with the results of bimolecular complementation studies (2), are consistent with HN and F promoting fusion by the provocateur (2) mechanism, which asserts that the HN-F interaction is triggered at the surface by receptor binding. Takimoto et al. (20) identified several mutations in the dimer interface of NDV HN that severely compromised fusion promotion. Subsequently, we showed that the fusion deficiency resulting from many of these mutations in the membraneproximal region of the interface, e.g., F220A and -N and S222A, -K, -N, and -T, correlated with impaired hemadsorption (HAd) activity at 37°C, despite wild-type (wt) NA activity (3). Modeling of this domain indicated that these mutations disrupted intermonomeric hydrogen bonds critical to the integrity of the dimer interface (3).A second sialic acid binding site (site II) was subsequently identified at the membrane-distal end of the interface that is composed of residues from both monomers (21) and is proposed (1) to maintain the target membrane in close proximity during fusion. Here, we test the hypothe...

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…The neuraminidase activity was reduced in the presence of zanamivir for all of the expressed viral chimeric proteins, indicating that zanamivir effectively interacts with catalytic site I. Figure 3C and 3D show that receptor binding activity is maintained despite zanamivir's blockade of site I (as evidenced by the inhibition of neuraminidase), indicating that the NDV site II was activated for receptor binding in the chimeric proteins as described for NDV AV HN wt and other G-HN and HN-HN chimeric proteins (25)(26)(27).…”

“…The analyses of NDV revealed two sialic acid binding regions, sites I and II, on HN. We previously reported that site II can be activated for receptor binding by small molecules (e.g., zanamivir) that occupy site I (25), and this finding was supported by recent analysis of a series of NDV HN mutants (25)(26)(27).…”

Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein

“…The globular domain of Nipah G starts at residue 189, therefore we incorporated the G stalk up to and including residue 188. The advantage of using the globular head of NDV HN (starting at NDV residue 124) is that the engagement of NDV HN binding site I by zanamivir leads to the activation of binding site II (38,53). The second binding site binds receptor more avidly but lacks receptorcleaving activity, and therefore the zanamivir-treated NDV HN is constitutively receptor engaged (53).…”

The Second Receptor Binding Site of the Globular Head of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Activates the Stalk of Multiple Paramyxovirus Receptor Binding Proteins To Trigger Fusion

“…It has recently been shown that NDV HNs with mutations in the NDV HN dimer interface, which led to decreased receptor binding, also impaired fusion promotion (13). Adding zanamivir to one of these HN mutants restored binding, suggesting that zanamivir binding to site I induced activation of site II in this mutant (33). In the present study, we demonstrate that receptor binding to NDV HN site II efficiently transmits the fusion signal to the stalk regions of not only NDV HN but also HPIV3 and Nipah virus (NiV) receptor binding proteins (HN and G).…”