Ronald M. Iorio

Amino Acid Substitutions in the F-Specific Domain in the Stalk of the Newcastle Disease Virus HN Protein Modulate Fusion and Interfere with Its Interaction with the F Protein

ABSTRACT The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus mediates attachment to sialic acid receptors, as well as cleavage of the same moiety. HN also interacts with the other viral glycoprotein, the fusion (F) protein, to promote membrane fusion. The ectodomain of the HN spike consists of a stalk and a terminal globular head. The most conserved part of the stalk consists of two heptad repeats separated by a nonhelical intervening region (residues 89 to 95). Several amino acid substitutions for a completely conserved proline residue in this region not only impair fusion and the HN-F interaction but also decrease neuraminidase activity in the globular domain, suggesting that the substitutions may alter HN structure. Substitutions for L94 also interfere with fusion and the HN-F interaction but have no significant effect on any other HN function. Amino acid substitutions at other positions in the intervening region also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the intervening region is critical to the role of HN in the promotion of fusion and may be directly involved in its interaction with the homologous F protein.

Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion.

ABSTRACT Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.

Functional and neutralization profile of seven overlapping antigenic sites on the HN glycoprotein of Newcastle disease virus: monoclonal antibodies to some sites prevent viral attachment

We have previously identified five antigenic sites on the hemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria isolate of Newcastle disease virus (Iorio and Bratt, J. Virol. 48, 440-450; Iorio et al., J. Gen. Virol. 67, 1393-1403). Two additional sites (designated 12 and 23) are now described, bringing to a total of seven the number of antigenic sites defined by our panel of neutralizing anti-HN antibodies. Competition antibody binding and additive neutralization assays reveal that each of these newly-identified sites overlaps two previously-defined ones. The seven HN antigenic sites thus form a continuum in the three-dimensional conformation of the molecule. Studies on the inhibition of hemagglutination (HA), neuraminidase (NA) and the attachment of virus to chick cell monolayers have been used to construct a functional profile of each antigenic site. Monoclonal antibodies (mAbs) to three overlapping sites (12, 2 and 23) inhibit HA and NA and prevent viral attachment to chick cell monolayers. These findings are consistent with the domains recognized by these mAbs being close to the NA and receptor-binding sites. MAbs to two other overlapping sites, 14 and 1 (which in turn, overlap site 12), inhibit HA quite effectively, and attachment to a lesser extent. Sites 14 and 1 probably identify a second domain involved in receptor recognition. MAbs to the two remaining sites (3 and 4), though neutralizing, are negative in all three assays, thus recognizing domains not involved in HA or NA or attachment to chick cells.

Mutations in the stalk of the measles virus hemagglutinin protein decrease fusion but do not interfere with virus-specific interaction with the homologous fusion protein.

ABSTRACT The hemagglutinin (H) protein of measles virus (MV) mediates attachment to cellular receptors. The ectodomain of the H spike is thought to consist of a membrane-proximal stalk and terminal globular head, in which resides the receptor-binding activity. Like other paramyxovirus attachment proteins, MV H also plays a role in fusion promotion, which is mediated through an interaction with the viral fusion (F) protein. The stalk of the hemagglutinin-neuraminidase (HN) protein of several paramyxoviruses determines specificity for the homologous F protein. In addition, mutations in a conserved domain in the Newcastle disease virus (NDV) HN stalk result in a sharp decrease in fusion and an impaired ability to interact with NDV F in a cell surface coimmunoprecipitation (co-IP) assay. The region of MV H that determines specificity for the F protein has not been identified. Here, we have adapted the co-IP assay to detect the MV H-F complex at the surface of transfected HeLa cells. We have also identified mutations in a domain in the MV H stalk, similar to the one in the NDV HN stalk, that also drastically reduce fusion yet do not block complex formation with MV F. These results indicate that this domain in the MV H stalk is required for fusion but suggest either that mutation of it indirectly affects the H-dependent activation of F or that the MV H-F interaction is mediated by more than one domain in H. This points to an apparent difference in the way the MV and NDV glycoproteins interact to regulate fusion.

Structural and Functional Relationship between the Receptor Recognition and Neuraminidase Activities of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Receptor Recognition Is Dependent on Neuraminidase Activity

ABSTRACT The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.

Structural and Mechanistic Studies of Measles Virus Illuminate Paramyxovirus Entry

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.

Fusion Deficiency Induced by Mutations at the Dimer Interface in the Newcastle Disease Virus Hemagglutinin-Neuraminidase Is due to a Temperature-Dependent Defect in Receptor Binding

ABSTRACT The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37°C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.

Paramyxoviruses: different receptors – different mechanisms of fusion

Paramyxovirus-mediated membrane fusion usually requires an interaction between the viral-attachment and -fusion proteins. The mechanism by which this interaction regulates fusion differs between paramyxoviruses that bind to sialic acid-containing receptors and those that recognize specific proteins. The recently solved structure of the globular head of the measles virus hemagglutinin suggests that this difference might be related to the location of the receptor-binding sites on the attachment proteins of the two classes of paramyxoviruses.

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.

Genetic variation within a neutralizing domain on the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

Previously, a panel of monoclonal antibodies recognizing epitopes in four antigenic sites on the haemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria strain of Newcastle disease virus were used in strain comparisons. Epitopes in three sites were found to be conserved while the epitope recognized by the single antibody to site 3 was not. A new panel of antibodies is described, two of which bind to epitopes in site 3 and six of which bind to a site (site 1,4) that overlaps with sites 1 and 4 as determined by analyses of variants, temperature-sensitive mutants, and strains by assays of neutralization of infectivity and binding in a radioimmunoassay. Neutralization of heterologous strains with the panel of antibodies revealed that both new site 3 epitopes are also highly divergent, while three additional epitopes outside site 3 (those in site 1,4) are highly conserved. The new site 3 antibodies can bind to virions of several heterologous strains without neutralizing infectivity. Thus, of the 10 epitopes we have now examined, all of three in site 3 are specific with respect to neutralization of infectivity for the homologous strain, while all of seven in other sites are conserved in heterologous strains. This suggests that the strain specificity originally described for a single site 3 epitope is, instead, a property of a much more extensive, poorly conserved domain on the HN molecule.