Mutated Form of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Interacts with the Homologous Fusion Protein despite Deficiencies in both Receptor Recognition and Fusion Promotion

Abstract

The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) is a multifunctional, type II membrane glycoprotein. It exists at the surface of virions and virus-infected cells as a tetrameric spike structure, consisting of a long, membrane-proximal stalk region that supports a terminal globular domain. The latter region of the spike is responsible for HNЈs recognition of sialic acid-containing receptors as well as its neuraminidase (NA) activity, which cleaves sialic acid from both soluble and membrane-bound glycoconjugates (18). All of the antibody binding sites on the protein also reside in the globular domain (21).Several hundred strains of NDV have been isolated from all over the world. The X-ray crystallographic structures of the globular domain of the HN from one of these, the Kansas strain, as well as the protein complexed with sialic acid or an inhibitor of NA, have been solved (3). These structures suggest that the attachment and NA activities of HN are mediated by a single sialic acid binding site in two different conformations. Consistent with this, substitutions for several active site residues abolish both attachment and NA (3).Similarly, substitutions for residues in the NA active site of the HN from the Australia-Victoria (AV) isolate of NDV (NDV-AV) also result in the loss of detectable attachment and NA (11). These substitutions include I175E, D198R, K236R, Y526L, and E547Q. However, our laboratory has shown that the attachment activity of each of these receptor recognitiondeficient mutants can be partially rescued by providing NA activity (11). Clearly, this is inconsistent with the existence of a single site for both attachment and NA. Rather, it suggests that the defi...

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

“…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.…”

“…1C). As a negative control, HN carrying a K236R mutation in site I fails to co-IP with F, as previously demonstrated (14). We have previously identified mutations for residues A89, L90, and L94 in the stalk of NDV HN, which modulate proportionately fusion and HN-F complex formation, consistent with this domain mediating the interaction with F (15).…”

Addition of N-Glycans in the Stalk of the Newcastle Disease Virus HN Protein Blocks Its Interaction with the F Protein and Prevents Fusion

“…The abilities of wt and mutated HN proteins to interact with cleavage site-mutated (csm) F at the surfaces of transfected BHK cells were assayed at 16 h posttransfection using a previously described co-IP assay (26).…”

Decreased Dependence on Receptor Recognition for the Fusion Promotion Activity of L289A-Mutated Newcastle Disease Virus Fusion Protein Correlates with a Monoclonal Antibody-Detected Conformational Change

“…The ability of HN to interact with F and L289A-F at the surface of transfected BHK cells was assayed at 16 h posttransfection, using a co-IP assay described previously (21). Briefly, monolayers consisting of equal numbers of cells were starved and labeled, prior to biotinylation of cell surface proteins.…”

“…This contribution is virus specific, in that most heterologous combinations of HN and F do not result in fusion (20). The role that HN plays in Fmediated fusion involves an interaction between the two proteins at the cell surface (7,21,33). Through the construction and evaluation of chimeric HN proteins composed of domains from proteins derived from heterologous viruses, it has been shown that the stalk region of the HN spike determines its specificity for the homologous F protein (8,29,31,32).…”