Paramyxoviruses are enveloped, negative-stranded RNA viruses characterized by the ability to form large multinucleate syncytia. The family Paramyxoviridae
includes mumps, measles, Sendai, respiratory syncytial, and Newcastle disease (NDV) viruses, as well as the various parainfluenza viruses (24
). Decorating the surfaces of NDV virions and infected cells are the hemagglutinin-neuraminidase (HN) and fusion (F) glycoprotein spikes (41
). The HN protein is responsible for binding to sialic acid-containing cellular receptors and, via its neuraminidase (NA) activity, cleavage of sialic acid from a number of different moieties, including these same receptors. The F protein is the mediator of virus-cell and cell-cell fusion.
HN is a type II membrane glycoprotein that exists on virion and infected-cell surfaces as a tetrameric spike (4
). The ectodomain of HN consists of a stalk region that supports a terminal globular head, in which reside the NA and attachment activities (37
), as well as all seven antigenic sites recognized by a panel of monoclonal antibodies (MAbs) (17
). The X-ray crystal structure of the head region of the HN protein from the Kansas strain of NDV has been solved (7
). An NA active site, capable of binding and releasing sialic acid analogues, was identified in each monomer. Though it was originally thought that this was the only sialic acid binding site in HN, the same group subsequently identified another site composed of residues from each monomer at the dimer interface (59
). This site is capable of binding to sialic acid analogues but lacks enzymatic activity.
F is a type I glycoprotein that exists on the surfaces of virions and infected cells as a homotrimeric spike (40
). It is synthesized as a precursor, F0
, which is cleaved into disulfide-linked polypeptides, F1
. At the new amino terminus of F1
is the fusion peptide (41
), which is inserted into the target membrane, thereby disordering the lipid bilayer and preparing it for fusion (24
For most paramyxoviruses, the F protein alone cannot mediate fusion; it requires coexpression of the homologous attachment protein (reviewed in reference 24
). Several different laboratories, including our own, have evaluated the fusion specificities of chimeric HN proteins with segments derived from heterologous paramyxoviruses (8
). All the data from these studies are consistent with the stalk region of HN being the site of the domain that determines F protein specificity, though this does not necessarily mean that it is the site of the domain that actually mediates the interaction with F.
Though the stalk region was not part of the HN crystal structure, other approaches have identified features of the region that are important for fusion. Some strains of NDV, including Australia-Victoria (NDV-AV), have a cysteine residue at position 123, which is involved in an intermonomeric disulfide bond (37
). Site-directed mutagenesis has demonstrated that the presence of a cysteine at this position increases the fusion-promoting activity of HN (7
). Also, within a conserved region (amino acids 74 to 110) are two small amphipathic α-helical motifs. These have been termed heptad repeats (HRs) (43
), although they do not adhere strictly to the aH-bP-cP-dH-eP-fP-gP (H, hydrophobic; P, polar) rule (12
) and are not predicted by tertiary-structure programs to form coiled coils (2
). Nonetheless, in keeping with convention, these motifs will be referred to here as HR1 and HR2.
Mutation of the heptadic residues in the “a” positions of HR1 (residues 74, 81, and 88) and HR2 (residues 96, 103, and 110) diminishes the fusion promotion activity of HN to 8 to 31% of wild type (wt), but most of these mutations also decrease the NA activity of the protein in the globular-head region (43
). Though this is consistent with the idea that this is the F-specific region on HN, one cannot rule out the possibility that mutations in the stalk that modulate fusion do so by affecting a domain in the head region, especially since NA activity resides there.
HR1 and HR2 are separated by a 7-amino-acid intervening region. We have recently shown that substitutions for some residues in this region interfere with fusion with no detectable effect on attachment or NA activity (35
). Moreover, diminished fusion by these mutants correlates with prevention of formation of the HN-F complex, as determined by a cell surface coimmunoprecipitation (co-IP) assay. These findings are consistent with this region directly mediating the interaction with F.
However, a peptide-based approach yielded a different result. Based on the assumption that HR-B in F mediates the interaction with HN and that a peptide mimicking this domain will bind specifically to a peptide containing the F-interactive domain on HN, a 20-mer peptide spanning the NDV F HR-B was tested for its ability to bind to peptides from various segments of NDV HN (15
). The HR-B peptide bound to a peptide mimicking amino acids 124 to 152 from HN, leading the authors to conclude that this is the site on HN that interacts with F. This was consistent with the finding that an HR-B peptide from the Sendai virus F protein was capable of binding to a soluble, stalkless form of HN (48
). Thus, despite a great deal of effort, some doubt still remains as to the determinants of the HN-F interaction on the two proteins.
The addition of N-glycans to viral glycoproteins has often been used to investigate the role of selected domains in protein function (1
). For example, Gallagher et al. (14
) showed that “supernumerary oligosaccharides” added to the influenza HA protein mask functional epitopes by shielding specific areas on the surface of the protein. Thus, the addition of N-glycans offers a straightforward approach to explore the role of a relatively large area in the function(s) of a protein.
NDV HN has six potential N-linked glycosylation sites, only four of which are utilized. One is in the stalk region at residue 119 (G1), and three are in the globular head at residues 341, 433, and 481 (G2, G3, and G4, respectively) (34
). However, two variant viruses with mutations of either D287N or K356N, selected with MAbs to antigenic sites 3 and 4, respectively, escape neutralization by the introduction of additional N-glycans (21
). Although neither of these N-glycans affects either the attachment or the NA activity of HN, the D287N mutant exhibits markedly reduced ability to promote fusion from within, the mode of fusion promoted by the viral glycoproteins on the cell surface, and unlike the parent virus, has acquired the ability to promote fusion from without, the mode of fusion directly mediated by input virions at high multiplicity (9
). Thus, the introduction of supernumerary oligosaccharides can affect the fusion properties of HN. These results led us to use this approach to explore the role in fusion of the HN stalk and the domain identified in the peptide-based approach.
Our results show that N-glycans added at either of two positions in the stalk of HN modulate only the HN-F interaction and fusion promotion function of HN with no detectable effect on the hemadsorption (HAd) or NA activity in the globular domain. These findings strongly support the idea that the stalk region of HN is directly involved in fusion promotion by mediating an interaction with the homologous F protein. N-glycans at other positions in the stalk similarly block fusion but also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as shown by sucrose gradient sedimentation analyses. Finally, N-glycan addition at residue 143 in a domain predicted by the peptide studies to mediate the interaction with F resulted in a quite significant level of fusion, arguing strongly against this site being part of the F-interactive domain in HN.