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Journal of Virology, January 2006, p. 623-633, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.623-633.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Vanessa R. Melanson and Ronald M. Iorio*

Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122

Received 19 August 2005/ Accepted 21 October 2005


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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.


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INTRODUCTION
 
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, 37, 38, 46). The ectodomain of HN consists of a stalk region that supports a terminal globular head, in which reside the NA and attachment activities (37, 47), as well as all seven antigenic sites recognized by a panel of monoclonal antibodies (MAbs) (17, 18, 20, 22). 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 and F2. 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, 11, 44, 51, 53). 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, 42). Site-directed mutagenesis has demonstrated that the presence of a cysteine at this position increases the fusion-promoting activity of HN (7, 33). Also, within a conserved region (amino acids 74 to 110) are two small amphipathic {alpha}-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, 16, 27, 32) and are not predicted by tertiary-structure programs to form coiled coils (2, 28, 56). 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, 52). 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, 14, 49, 54). 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.


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MATERIALS AND METHODS
 
Recombinant plasmids and site-directed mutagenesis. Construction of NDV-AV HN and F recombinant pBluescript SK(+) (Stratagene Cloning Systems, La Jolla, CA) expression vectors (37) and preparation of site-directed mutants were performed as previously described (35).

Transient-expression system and quantitation of viral protein expression. Wt and mutated HN proteins were expressed in BHK-21 cells (American Type Culture Collection, Manassas, VA) using the vaccinia virus T7 RNA polymerase expression system (13). All experiments, except the NA assays, were performed on 35-mm plates seeded a day earlier at 4 x 105 cells per well. Maintenance of cells, infection with recombinant vaccinia virus vTF7-3, and transfection were performed as described previously (37), using 1 µg of each plasmid for transfection. Cell surface expression (CSE) was quantitated by flow cytometric analysis with a mixture of MAbs to at least five different antigenic sites in the HN globular-head domain (17, 18, 20, 22).

Functional assays. HAd activity was determined by the abilities of the expressed HN proteins to adsorb guinea pig erythrocytes (Bio-Link, Inc., Liverpool, NY) (35). The NA activity of cell surface HN was determined by the abilities of the expressed proteins to enzymatically process the substrate neuraminlactose (Sigma Chemical Co., St. Louis, MO) (35). The abilities of the mutated HN proteins to complement the F protein in the promotion of fusion were quantitated using a content-mixing assay, which measures ß-galactosidase activity in target cells following fusion induced by HN-F-expressing effector cells (35).

IP and peptidyl-N-glycosidase (PNGase) F digestion. The immunoprecipitation (IP) protocol was described previously (25). Briefly, at 22 h posttransfection, BHK cells were starved for 1 h at 37°C in medium lacking cysteine and methionine. The cells were labeled with 1 ml of medium containing 100 µCi of Expre35S [35S]cysteine-methionine labeling mix (Dupont-New England Nuclear, Boston, MA) for 3 h at 37°C, followed by a 90-min chase with medium. The cells were lysed, and the HN proteins were immunoprecipitated with a cocktail of five MAbs. The antigen-antibody complexes were collected with Ultralink-Immobilized Protein A Plus (Pierce, Rockford, IL) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

For PNGase F digestion, the Ultralink-Immobilized Protein A Plus with bound immunoprecipitate was resuspended in PNGase buffer (0.1 M sodium phosphate [pH 7.2], 25 mM EDTA) containing 0.8% SDS and boiled for 5 min. The solution was allowed to cool and adjusted to contain 0.1% SDS and 0.5% NP-40 in the above-mentioned buffer. One aliquot of each sample was digested with 200 mU of PNGase F (New England Biolabs, Beverly, MA) for 16 h at 37°C prior to SDS-PAGE under reducing conditions (9).

Co-IP assay. 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).

Sucrose gradient sedimentation analyses. Cells expressing wt and mutated HN proteins were lysed for 30 min with 20 mM morpholino-ethanesulfonic acid, 30 mM Tris, 100 mM NaCl (MNT buffer), pH 5.0 (38), containing 0.5% dodecyl-ß-D-maltoside (DM), 20 mM iodoacetamide, and 1% phenylmethylsulfonyl fluoride. Cell debris and nuclei were removed by centrifugation for 5 min, and 0.5 ml of each supernatant was layered onto continuous gradients of 7.5 to 22.5% sucrose in MNT buffer plus 0.05% DM with a 0.5-ml pad of 65% sucrose. The gradients were centrifuged at 37,000 rpm for 16 h at 19°C in a model SW41 Beckman-Coulter rotor. Fractions (350 µl) were collected, and proteins in aliquots from alternate fractions were precipitated with trichloroacetic acid and displayed by SDS-PAGE under nonreducing conditions. Molecular mass markers were bovine albumin (67 kDa), aldolase (160 kDa), catalase (240 kDa), and ferritin (450 kDa) (Crescent Chemical Company, Inc., Islandia, NY), the locations of which in the gradients were detected by Coomassie brilliant blue staining. Wt and mutated HN proteins were electroblotted onto Immobilon P membranes (Millipore Corp.) for 18 h at 100 mA for Western analysis.

Western blots. Membranes were blocked for 1 h with 5% nonfat milk in phosphate-buffered saline containing 0.5% Tween 20 and incubated for 1 h with hybridoma supernatant containing an antibody to antigenic site 14, which recognizes a linear epitope in HN (22). The membranes were then incubated for 1 h with a 1/2,500 dilution of horseradish peroxidase-conjugated goat anti-mouse antibody (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). All incubations were done on a rocking platform at room temperature, and the membranes were washed repeatedly with phosphate-buffered saline containing 0.5% Tween 20 between incubations. Antibody binding was detected using the ECL Western Blotting Detection system (Amersham Biosciences, Piscataway, NJ) according to protocols provided by the manufacturer.


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RESULTS
 
N-glycans added at either of two positions in the HN stalk interfere with fusion promotion. Earlier studies are consistent with the stalk of NDV HN playing a role in the HN-F interaction that is required for fusion (11, 35). Here, we used the addition of N-glycans at various positions in this region as an alternate approach to further explore its role in the HN-F interaction and fusion. N-linked carbohydrates are covalently attached to asparagines on nascent polypeptides at the motif N-X-T/S, where X is any amino acid except aspartic acid or proline (23).

Initially, N-glycans were introduced to "shield" specific parts of HR1. The sites of N-glycan addition were chosen to minimize sequence changes in the region. Specifically, two potential N-linked glycosylation sites were introduced at the third and ninth residues from the HR1-initiating leucine residue (L74): D79T (N-glycan addition at N77) and R83N plus Y85S. Prior to a functional analysis of the mutated HN proteins, expression of each was confirmed by flow cytometry with a panel of MAbs specific for at least four antigenic sites on HN. As shown in Fig. 1A, these mutated HN proteins are expressed at greater than 90% relative to wt HN. This verifies not only that they are efficiently expressed, but also that neither of them is misfolded.


Figure 1
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  		FIG. 1. Substitutions D79T and R83N plus Y85S add N-glycans to NDV AV-HN and variously affect the functions of HN. (A) CSE is determined by flow cytometry using a cocktail of five anti-HN MAbs specific for different sites on the globular head of the NDV AV-HN protein. HAd activity is determined by the ability of the expressed HN proteins to adsorb guinea pig erythrocytes. NA activity is determined by the ability of the expressed HN proteins to catalyze the release of sialic acid from neuraminlactose. The NA data were corrected for differences in expression. The fusion promotion activity of the HN proteins was determined by the content-mixing assay. Both D79T-HN and R83N plus Y85S-HN exhibited no detectable fusion promotion activity. Thus, there is no bar on the graph for these mutated HN proteins. For all four of these assays, the background level obtained with vector alone was subtracted. All data are expressed relative to the amount of the wt HN protein and represent the means of at least four independent determinations plus standard deviations. (B) BHK cells were transfected with pBluescript SK(+) vector alone, wt HN, D79T-HN, or R83N plus Y85S-HN. The cells were labeled and lysed, and the lysate was divided into two aliquots prior to immunoprecipitation of HN. After being washed, the immunoprecipitates were resuspended in PNGase F buffer. One aliquot was digested with 200 mU of PNGase F, and both aliquots were electrophoresed under reducing conditions.

To determine whether the introduced N-linked glycosylation sites are utilized, immunoprecipitation of each radiolabeled mutated protein with a panel of MAbs to HN was performed. Figure 1B shows that the two mutated proteins migrate at a lower rate in the gel than wt HN, consistent with the addition of an N-glycan. That this is indeed the reason for the slower migration of the mutated HN proteins was verified by treatment of the immunoprecipitates with PNGase F. This enzyme cleaves the N-glycan linkage of glycoproteins between asparagine and the carbohydrate chain (45). After digestion with PNGase F, the mutated proteins comigrate with wt HN at a higher rate, confirming that the altered migration rate of the mutated HN proteins is due solely to a difference in N-linked glycosylation.

Next, the effects of the additional N-glycans on the ability of HN to complement F in the promotion of fusion were determined by a content-mixing assay. Neither D79T-HN nor R83N plus Y85S-HN is able to promote a detectable level of fusion (Fig. 1A). Therefore, the addition of an N-glycan at either position abolishes the fusion promotion activity of HN.

There is a possibility that the diminished ability of the mutants to promote fusion is related to an alteration in their ability to bind to cellular receptors. Thus, the receptor binding activities of the two mutated proteins were examined with an HAd assay. As shown in Fig. 1A, both D79T-HN and R83N plus Y85S-HN display wt levels of HAd. Therefore, their lack of fusion promotion is not due to diminished binding to receptors.

Another function of HN that has been known to modulate fusion promotion is NA activity, most likely due to its ability to release HN from sialic acid-containing receptors (39). Indeed, R83N plus Y85S-HN displays severely diminished NA activity, 11% of that of wt HN (Fig. 1A). Although receptor recognition is dependent on NA activity, this mutated HN protein exhibits sufficient NA activity to maintain wt levels of attachment (36). Interestingly, D79T-HN has wt NA activity. Thus, the fusion deficiency of D79T-HN is not related to a change in any other known HN function; both attachment and NA activities are similar to those of wt HN.

Since the N-glycan added at N77 via the D79T substitution affected only fusion and the N-glycan introduced by the R83N plus Y85S substitution affected both fusion promotion and NA, we introduced another potential N-linked glycosylation site into the HN stalk at residue 69 (K69N substitution), which is membrane proximal from HR1, to determine if an N-glycan at this position has the same phenotype as D79T. Through analysis by SDS-PAGE and digestion with PNGase F, we determined that K69N-HN is glycosylated. The mutated protein migrates at a lower rate than wt HN, and following digestion with PNGase F, both wt and K69N-HN comigrate at a higher rate, confirming that the altered migration rate of the untreated mutated protein is due to a difference in glycosylation (Fig. 2A). Flow cytometry with a panel of anti-HN MAbs established the expression level of K69N-HN to be similar to that of wt HN (92.4%) (Fig. 2B). The fusion promotion, receptor recognition, and NA activities of K69N-HN were then tested. Figure 2B shows that this mutated protein promotes approximately 3% of wt fusion, 94% of wt HAd, and 94% of wt NA. Therefore, analogous to D79T-HN, N-glycan addition at K69N in the HN stalk specifically decreases only the fusion promotion activity of HN, with no detectable effect on its other activities. Thus, N-glycan additions at two different sites in the HN stalk result in proteins that maintain all the functions of HN except its fusion promotion activity.


Figure 2
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  		FIG. 2. A K69N substitution adds an N-glycan to NDV AV-HN and affects only the fusion promotion of the protein. (A) The IP assay and the PNGase F treatment were performed as described in the legend to Fig. 1. (B) The functional assays were performed as described in the legend to Fig. 1. The data represent the means of at least four independent determinations plus standard deviations.

To determine whether the diminished fusion of K69N-HN and D79T-HN can be induced merely by amino acid substitutions at those positions or whether it is due to the presence of the N-glycan, we expressed HN proteins carrying each of the following substitutions: K69A, N77A, D79E, D79L, or D79R. Figure 3 shows that all of these mutated HN proteins are expressed at wt levels and display wt levels of receptor recognition, NA, and fusion promotion activities. Therefore, none of these substitutions significantly alters any HN function, suggesting that it is the N-glycan rather than the amino acid substitution itself that is responsible for the fusion defect exhibited by each mutated protein.


Figure 3
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  		FIG. 3. CSE and functional assays for HN proteins carrying substitutions for residues K69, N77, D79, R83, and Y85. The assays were performed as described in the legend to Fig. 1. The data represent the means of at least four independent determinations plus standard deviations.

An R83N substitution specifically affects fusion. Unlike K69N-HN and D79T-HN, R83N plus Y85S-HN exhibits not only fusion deficiency, but also markedly reduced NA activity (Fig. 1A). Since this is a double mutant, we determined the contribution of each substitution to this phenotype. Both R83N-HN and Y85S-HN are expressed at wt levels and exhibit wt receptor binding activity (see Fig. 3). Both mutated HN proteins reduce fusion promotion to less than 20% of the wt amount. However, Y85S-HN also reduces NA activity by approximately 50%, while R83N-HN displays wt levels. Therefore, the R83N substitution affects only fusion without modulating the other activities of HN.

Correlation between fusion deficiency and interference with the HN-F interaction. To determine if K69N-HN and D79T-HN, which have added N-glycans, and R83N-HN, which does not, affect fusion promotion by interfering with the HN-F interaction, a co-IP assay was performed with each mutated HN protein. As shown in Fig. 4, both overglycosylated proteins, K69N-HN and D79T-HN, are unable to interact in detectable amounts with the F protein. This correlates with their lack of ability to promote significant amounts of fusion. As controls, K69A-HN, N77A-HN, and D79E-HN, which promote wt levels of fusion, interact efficiently with wt F.


Figure 4
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  		FIG. 4. Co-IP of NDV AV-HN and K69-, N77-, and D79-mutated HNs with csm F. Equal numbers of cells were transfected, as indicated. After 16 h, the cells were starved and labeled. The cell surface proteins were biotinylated, and the cells were lysed in DM. The lysate was split into two aliquots and immunoprecipitated with either a combination of an anti-F MAb and a cocktail of anti-HN MAbs (the first lane in each pair) or an anti-F MAb alone (the second lane in each pair). The immunoprecipitates were collected with protein A-agarose and washed before being boiled in SDS and reprecipitation with strepavidin agarose prior to analysis by SDS-PAGE. The F protein exhibits a faster and sharper migration pattern in the presence of HN due to the trimming of sialic acid from the N-glycans on F by the NA activity of HN (10, 57).

The co-IP assay for R83N-HN, Y85S-HN, and R83N plus Y85S-HN is shown in Fig. 5. Again, none of the mutated HN proteins interacts with the F protein, consistent with their inability to promote fusion greater than 20% of the wt amount. We have previously shown that the detection limit of the co-IP assay corresponds to approximately 20% of wt fusion (35).


Figure 5
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  		FIG. 5. Co-IP of NDV AV-HN and R83- and Y85-mutated HNs with csm F. The experiment was performed as described in the legend to Fig. 4.

Addition of N-glycans in HR2 also blocks fusion. Next, we examined HR2, which is more conserved among paramyxovirus HN proteins, and hence, less likely to be directly involved in the virus-specific HN-F interaction. Potential N-linked glycosylation sites were introduced at the third through the ninth residues from the initiating leucine residue (L96) in HR2. In this way, the effect on HN function of glycosylation at each position in the conserved {alpha}-helical structure of HR2 can be determined. Thus, the following amino acid substitutions were introduced: T99N, E100N plus I102S, S101N plus I103T, I102N plus M104T, I103N plus N105S, M104N plus A106T, I107S (glycan addition at N105), and A106N. All of these mutated HN proteins are indeed glycosylated, as evidenced by comparison of their migration rates on SDS-PAGE before (Fig. 6A) and after (Fig. 6B) PNGase F digestion. In addition, their expression levels, as determined by flow cytometry, range from 93 to 107% of the wt level (Fig. 7).


Figure 6
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  		FIG. 6. Substitutions introduced into HR2 of the NDV AV-HN stalk all result in the addition of N-glycans. (A) The IP assay was performed as described in the legend to Fig. 1. Two wt HN bands are shown to highlight the retarded migration of the N-glycan addition mutants. (B) The PNGase F treatment was performed as described in the legend to Fig. 1.


Figure 7
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  		FIG. 7. CSE and functional assays of mutated HN proteins carrying supernumerary N-glycans in HR2. The assays were performed as described in the legend to Fig. 1. Only I102N plus M104T-HN and I107S-HN exhibited extremely low levels of fusion. The other six mutations completely abolished fusion; therefore, this phenotype registers as no bar on the graph. The data represent the means of at least four independent determinations plus standard deviations.

As shown in Fig. 7, most of these mutated HN proteins are unable to promote a detectable level of fusion. The mutated protein I107S-HN exhibits barely detectable fusion activity, promoting less than 2% of wt HN fusion. This amount of fusion, though detectable in the content-mixing assay, is too weak to be visible in cellular monolayers (data not shown). Thus, N-glycan addition at any of several positions in HR2 eliminates fusion.

We then tested the receptor recognition and NA activities of the HR2 mutated proteins. As shown in Fig. 7, one-half of the mutated proteins, E100N plus I102S-HN, S101N plus I103T-HN, I103N plus N105S-HN, and M104N plus A106T-HN, have increased levels of HAd compared to the wt (134 to 150%), while the other half, T99N-HN, I102N plus M104T-HN, I107S-HN, and A106N-HN, have decreased levels (54 to 66%). These alterations in HAd activity are not significant enough to alter the level of fusion (6). However, all the HR2 N-glycan addition mutants do exhibit decreased NA activity, 7 to 18% of the wt level, though this amount of NA is still high enough to allow efficient recognition of receptors (36). However, the modulation of both the NA and HAd activities of these HR2 N-glycan addition mutants suggests that they may be altered structurally. However, the alteration is too slight to be detectable by the anti-HN MAbs.

Decreased NA activity correlates with an altered sedimentation profile in sucrose gradients. The stalk region of the HN spike is critical for stabilizing the structure of the tetramer in the absence of ligand (58). Thus, the addition of N-glycans in the stalk might reasonably be expected to alter the structure of the tetramer. To investigate this possibility, we probed for alterations in HN tetramer structure in the HR2-mutated proteins by sucrose gradient sedimentation analysis. Shown in Fig. 8 are sucrose gradient sedimentation profiles of wt HN and five mutated HN proteins selected on the basis of differences in the site of N-glycan addition and the effect it has on HN function. Two of the mutated proteins, K69N-HN (Fig. 8B) and D79T-HN (Fig. 8C), sediment in the gradient at a rate similar to that of the wt protein (Fig. 8A), with the majority of the protein in fractions 9 to 15. This suggests that the structure of the HN tetramer is not altered by N-glycan addition at these two positions. These results are consistent with the fact that both mutations affect only the HN-F interaction and fusion, as well as with this phenotype being due to a direct effect on the HN-F interaction, rather than to an alteration in the structure of the tetramer.


Figure 8
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  		FIG. 8. Sucrose gradient sedimentation analysis of mutated HN proteins carrying supernumerary N-glycans. Cells expressing wt and mutated HN proteins were lysed and layered onto continuous 7.5 to 22.5% sucrose gradients in MNT buffer plus 0.05% DM. The proteins in odd-numbered fractions were subjected to trichloroacetic acid precipitation, SDS-PAGE under nonreducing conditions, and Western blot analysis. Companion gradients were run with the molecular mass markers aldolase (160 kDa) and catalase (240 kDa), the sedimentation of which is indicated by the arrows at the top. Bands containing the monomeric (M), dimeric (D), and tetrameric (T) forms of HN in each gel are indicated, as are the tops and bottoms of the gradients. The lane on each gel containing the lysate is labeled "L." (A) wt HN; (B) K69N-HN; (C) D79T-HN; (D) E100N plus I102S-HN; (E) I107S-HN; (F) A106N-HN.

The sedimentation profiles of the remaining three mutated proteins, E100N plus I102S-HN (Fig. 8D), I107S-HN (N105) (Fig. 8E), and A106N-HN (Fig. 8F), exhibit a biphasic pattern with two distinct peaks, one in fractions 9 to 13 and a second in fractions 15 to 19 (Fig. 8). The second peak corresponds to the sedimentation rate of the 160-kDa marker, consistent with the size of the dimeric form of the protein. This sedimentation pattern indicates that the structure of the HN tetramer is altered by N-glycan addition at these positions. This is consistent with the fact that all three mutated proteins severely impair NA activity (Fig. 7), which resides in the globular domain. The effect on NA could be related to the fact that the NA activity of NDV-AV exhibits sigmoidal substrate saturation kinetics indicative of cooperativity (31). Thus, it would certainly not be surprising for changes in the association between the globular domains in the tetramer to affect NA activity.

A106N-HN (Fig. 8F) exhibits another interesting difference from the wt protein. In addition to sedimenting as a dimer and a tetramer, some of each oligomer runs on the nonreducing gel as a monomer. One possible explanation for this observation is that the introduction of the N-glycan at position 106, though maintaining the dimeric or tetrameric form, interferes with formation of the intermonomeric disulfide bond mediated by the cysteine at position 123 (42).

HN carrying an N-glycan at residue 143 retains a significant amount of its fusion-promoting activity. Based on peptide binding studies, it has been concluded that NDV HN residues 124 to 152 mediate the interaction with F (15). As an alternative to a detailed site-directed mutagenic analysis of the role of this domain in fusion, we introduced a single glycosylation site at a convenient position in the region. A D143N substitution introduces a site, which is utilized, as evidenced by a retarded migration rate on SDS-PAGE of the mutated protein relative to wt HN and comigration of the two proteins following PNGase F digestion (Fig. 9A). As shown in Fig. 9B, this mutation does not affect cell surface expression of the protein, and the mutated protein retains more than 60% of its fusion activity. The decrease in fusion is probably related to a decrease in HAd (38.1% of wt) and NA (15.1% of wt) activities. Thus, the presence of an N-glycan in the middle of this putative F-interactive domain still allows a quite significant level of fusion.


Figure 9
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  		FIG. 9. D143N-HN carries an additional N-glycan but still promotes a significant amount of fusion with the homologous F protein. (A) The IP assay and PNGase F treatment were performed as described in the legend to Fig. 1. (B) The functional assays were performed as described in the legend to Fig. 1. The data represent the means of at least four independent determinations plus standard deviations.


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DISCUSSION
 
Several aspects of the mechanism of HN-dependent F-mediated fusion still remain to be elucidated. Chief among these are the identities of the virus-specific interactive sites on the two proteins. Several approaches have been used to localize these domains. Chimeric HN and F proteins composed of domains from different paramyxoviruses have been evaluated for the ability to promote fusion. Analysis of several different chimeric HN proteins showed that specificity for the homologous F protein is determined by the stalk region of the HN spike (8, 11, 44, 51, 53). Similarly, analysis of chimeric F proteins indicated that specificity for the homologous HN/H protein is determined by the N-terminal half of the cysteine-rich domain in F and/or the heptad repeat closest to the membrane in the stalk of F (50, 55).

Based on the results of these studies, we previously determined the effects of point mutations in a semiconserved domain in the stalk of NDV HN on its ability to promote fusion and to interact with the homologous F protein. Mutations at position A89, L90, or L94 in the intervening region between two HRs in the HN stalk severely impair its ability to complement NDV F in the promotion of fusion (35). This fusion deficiency correlates with an inability to detect an interaction between the two proteins at the cell surface. These are the first point mutations identified in any HN protein for which a fusion-deficient phenotype has been shown to correlate with a loss of the ability to interact with F.

Many studies have used the approach of introducing additional N-glycans to surface glycoproteins to explore their effects on intracellular transport, surface expression, and function (1, 14, 29, 30, 49). This approach is particularly useful when additional functional assays are available to rule out global effects on the protein. The multifunctional nature of the paramyxovirus HN proteins makes it particularly amenable to this approach. Here, we introduced supernumerary N-glycans in key regions of the NDV HN protein that are implicated in its interaction with the homologous F protein that is necessary for fusion. N-glycans added at two positions in the HR region of the stalk, residues 69 and 77, specifically abolish the fusion-promoting activity of HN with no significant effect on its attachment or NA functions. This loss of fusion correlates with a loss of the ability to interact with the homologous F protein at the cell surface. These results are consistent with the chimera and site-directed mutagenesis data that point to the stalk region as the site of the F-interactive domain in NDV HN (11, 35).

Sucrose gradient analyses suggest that the tetrameric structure of the HN protein is unaltered by the addition of N-glycans at positions 69 and 77. This, coupled with the fact that no other function of HN is affected, is consistent with a mechanism in which the glycans block fusion by sterically interfering with the HN-F interaction. This argues strongly that this region is part of the F-interactive site on HN.

On the other hand, our data are inconsistent with the involvement of a domain defined by residues 124 to 152 in the interaction with F, as has been concluded from the results of a peptide-based approach (15). The introduction of an N-glycan via a D143N mutation does not eliminate fusion; indeed, the mutated protein retains more than 60% of its fusion-promoting activity. Since N-glycans are large hydrophilic structures, they would certainly be expected to interrupt any protein-protein interaction involving this domain.

In some ways, the latter result is not surprising. An interaction between HR-B in F and residues 124 to 152 in HN is difficult to justify for at least two reasons. First, NDV-human parainfluenza virus type 3 (hPIV3) HN chimeras that contain NDV HN residues 124 to 152 fuse with hPIV3 F, but not NDV F (11). Second, residues 124 to 152 in HN are much further from the membrane sequence than is HR-B in F. Thus, a bend in the HN stalk significantly greater than 90o would be required to bring together the proposed complementary domains on the two proteins. If the globular head of HN is down so close to the membrane in which the molecule resides, and especially beneath the F globular domain, it is difficult to envision how it can interact with receptors on the target cell surface or be accessible to neutralizing antibodies, all of which interact with sites in the globular head (37).

Based on the consensus secondary-structure prediction results from Network Protein Sequence Analysis (5), residues 69 and 77 lie in a region predicted to form a random coil (Fig. 10A). This may explain why N-glycans at these two positions do not alter the structure of the protein. In addition, residues 69 and 77 are further away from the globular head than any of the other N-glycan addition sites and therefore may not have such a drastic affect on HN's HAd and NA functions in the head.


Figure 10
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  		FIG. 10. (A) Amino acid sequence for the wt NDV AV-HN protein showing residues 65 to 125. HR1 and HR2 are indicated and labeled a to g. The heptadic residues in position "a" are in boldface. The intervening region (IR) is also indicated. A naturally occurring N-glycan at residue 119 ({triangleup}) and the cysteine involved in an intermonomeric disulfide bond at residue 123 (*) are also indicated. The secondary-structure prediction of the entire NDV AV-HN amino acid sequence was done on Network Protein Sequence Analysis (5). Eight secondary-structure prediction programs (DPM, DSC, GOR4, HNNC, PHD, Predator, SIMPA96, and SOPM) were selected for the analysis. Pluses denote the areas that are predicted to be {alpha}-helical by the consensus secondary-structure prediction results of the eight programs. Residues 69 and 77, at which N-glycans are added, are indicated by triangles. The amino acid substitutions responsible for the introduction of these two N-glycans are indicated. (B) Helical wheel diagram of HR2 residues 96 to 110 of NDV AV-HN. The most amino-terminal residue, L96, is placed at position "a" of the idealized {alpha}-helix. N-glycan addition to certain residues is indicated ({triangleup}). The dotted line represents the demarcation of the two faces of the helix, with {uparrow} HAd indicating an increase in hemadsorption and {downarrow} HAd indicating a decrease in hemadsorption.

N-glycan addition at position 83 (R83N plus Y85S) not only diminishes fusion and disrupts the HN-F interaction, but also severely reduces NA activity. By individually introducing each point mutation at positions 83 and 85, it was shown that the substitution at residue 85 is partially responsible for the decreased NA activity. However, the significant decrease in NA activity appears to be the result of a synergistic effect of the two mutations. However, each individual mutation decreases fusion and abolishes the interaction with F, so we cannot conclude that the N-glycan at position 83 is solely responsible for the decreased fusion and HN-F interaction phenotype. However, it is interesting that a single point mutation of R83N produces a phenotype similar to that of our previously identified L94 substitutions (35). Perhaps this residue is also directly involved in HN's interaction with F.

All the N-glycans added in HR2 almost completely abolish fusion and the HN-F interaction. However, each also significantly decreases NA activity, which suggests that N-glycans added in this region alter HN structure. Sucrose gradient sedimentation analyses of HR2-mutated proteins confirm this. Also, interestingly, HAd is affected differently depending on the site of N-glycan addition. Addition at residues T99, I102, N105, and A106 decreases HAd, while addition at residues E100, S101, I103, and M104 increases it (Fig. 10B). These two groups of residues are situated on opposite sides of the helix. On the side of the helix at which N-glycan addition decreases HAd, the N-glycan may be disrupting HN's second sialic acid binding site formed by the dimeric interface, thereby decreasing HN's receptor binding avidity. This idea is based on the demonstrated role of the second sialic acid binding site in attachment (3). N-glycan addition to the other side of the HR2 helix increases receptor binding avidity and may do so by stabilizing the receptor-binding structure of HN, resulting in a more efficient interaction with receptors.

In summary, we have shown that addition of N-glycans at any of several positions in the stalk of NDV HN abolishes its ability to complement the homologous F protein in the promotion of fusion and that this correlates with an inability to interact with the F protein at the cell surface. Two of these added N-glycans (at positions 69 and 77) specifically affect only fusion; attachment and NA, which reside in the head, are not affected. Several other added N-glycans abolish fusion but also affect HAd and NA. The latter phenotype correlates with an altered sedimentation pattern on sucrose gradients relative to that of the wt protein. Substitutions at positions 69 and 77, which affect only fusion, do not alter the sedimentation pattern. These results make a strong argument for the site of the F-interactive domain in HN residing in the stalk segment of the protein.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge Judith Alamares, Elizabeth Corey, Paul Mahon, Anne Mirza, and Mary Munson for helpful discussions. We also thank Robert Lamb and Trudy Morrison for the NDV F and HN genes, respectively, and Bernard Moss for the recombinant vaccinia virus.

This work was made possible by grant AI-49268 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0122. Phone: (508) 856-5257. Fax: (508) 856-5920. E-mail: Ronald.Iorio{at}umassmed.edu. Back


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Journal of Virology, January 2006, p. 623-633, Vol. 80, No. 2
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