JOURNAL OF VIROLOGY, Nov. 2008, p. 10386–10396
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 21
Engineered Intermonomeric Disulfide Bonds in the Globular Domain
of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein:
Implications for the Mechanism of Fusion Promotion?
Paul J. Mahon,1,2Anne M. Mirza,1Thomas A. Musich,3and Ronald M. Iorio1,3*
Department of Molecular Genetics and Microbiology1and Program in Immunology and Virology,3University of Massachusetts
Medical School, Worcester, Massachusetts 01655, and Department of Biology, Assumption College,
Worcester, Massachusetts 016092
Received 14 March 2008/Accepted 7 August 2008
The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the
viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this
interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of
dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat.
Struct. Biol. 7:1068–1074, 2000), it was proposed that the protein undergoes a significant conformational
change from an initial structure having minimal intermonomeric contacts to a structure with a much more
extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with
the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However,
no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test
the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the
globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substi-
tutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the
absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude
formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37°C
and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed
drastic conformational change in the protein is required for fusion.
The Paramyxoviridae are a family of enveloped, negative-
strand RNA viruses, which includes several important patho-
gens, such as measles virus, mumps virus, Sendai virus, respi-
ratory syncytial virus, the various parainfluenza viruses, and
Newcastle disease virus (NDV) (26). Although predominantly
recognized as an avian pathogen, NDV has recently gained
added importance for its ability to selectively kill tumor cells
and its use as an oncolytic agent (12, 25, 45), as well as its
potential as a vaccine vector (11, 14, 16, 29, 37).
The surfaces of paramyxovirions and infected cells possess
two types of spikes, composed of the attachment and fusion
proteins. For paramyxoviruses that recognize sialic acid-con-
taining receptors, the hemagglutinin-neuraminidase (HN) pro-
tein mediates receptor binding and also possesses sialidase or
neuraminidase (NA) activity, the ability to cleave sialic acid
(39). The fusion (F) protein mediates virus-cell and cell-cell
fusion for all paramyxoviruses (2), following the proteolytic
generation of a “fusion peptide” (39).
For most paramyxoviruses, including NDV, the F protein is
incapable of promoting fusion by itself (reviewed in reference
26). It requires a virus-specific contribution from the homolo-
gous attachment protein, which is mediated by a direct inter-
action between the two protein spikes. A complex between
NDV HN and F can be detected at the surface of both infected
and transfected cells (9, 28, 30–32). By the analysis of chimeric
HN proteins, it has been shown for several viruses in the
family, including NDV, that the stalk region of HN determines
its specificity for the homologous F protein (7, 10, 42, 44, 46).
Moreover, we have shown that the introduction of N-linked
glycans and point mutations in the HN stalk severely impairs,
or completely eliminates, fusion and that this correlates with a
proportionate decrease in the extent of HN-F complex forma-
tion at the cell surface (31, 32).
Like other paramyxovirus HN proteins (3, 36, 43), NDV HN
is a type II membrane glycoprotein that exists on the virion and
infected-cell surface as a tetramer comprised of a pair of
dimers (34). The ectodomain consists of a long stalk support-
ing a terminal globular domain, in which reside the attach-
ment, NA and antigenic sites (17–19, 23, 34, 43).
The X-ray crystal structures of the ligand-bound and unli-
ganded dimer of the globular domain of the NDV HN protein
have been determined (6). Each monomer has a ?-sheet pro-
peller motif with an NA active site at its center. Based on these
structures, it was postulated that the NA site mediates both
attachment and NA activity via a quite drastic conformational
change from a structure having a minimal dimer interface to
one with a much more extended interface (6). Further, it was
postulated that this drastic conformational change is integral
to HN?s role in the fusion process. Hydrophobic residues ex-
posed in the minimal interface form of the protein were pro-
posed to interact with complementary residues in the F pro-
tein, thus maintaining the latter in its prefusion conformation.
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, University of Massachusetts Medical
School, 55 Lake Avenue North, Worcester, MA 01655. Phone: (508)
856-5257. Fax: (508) 856-5920. E-mail: email@example.com.
?Published ahead of print on 27 August 2008.
Upon receptor binding, the protein was proposed to switch to
a markedly different structure, in the process sequestering the
purported F-interactive hydrophobic residues in a much more
extensive interface and releasing F to assume its fusion-active
form (6). This was supported by the claim that mutations of
some of the hydrophobic residues abolished fusion with no
effect on attachment (41). Subsequently, a second sialic acid
binding site was identified (48); it is positioned at the mem-
brane-distal end of the dimer interface, is composed of resi-
dues from both monomers, and lacks NA activity. This second
site was proposed to maintain the interaction of HN with
receptors as fusion proceeds (1).
However, this mechanism for fusion promotion is inconsis-
tent with the evidence cited above indicating that it is the stalk
region of NDV HN that determines its F specificity and may
directly mediate the interaction with F. The model was also
subsequently called into question by our demonstration that at
least some of the interface mutations that decrease fusion do
significantly affect attachment at 37°C, the temperature at
which fusion is assayed (5). Finally, the mechanism proposed
for the role of the NDV HN protein in fusion is not supported
by studies of two other paramyxoviruses. The structures of the
HN from human parainfluenza virus 3 (hPIV3) both unligan-
ded and with several ligands were determined at pH 7.5 (27).
Although the structure is similar to that of NDV HN, there is
a single flexible site that mediates both receptor binding and
NA by a structural change that is limited to the active site.
Similarly, the structure of the parainfluenza virus 5 (PIV5) HN
protein also identifies a single site with only a single flexible
tyrosine residue (47). There is no crystallographic evidence in
hPIV3 or PIV5 HN for either a second site or a conformational
change upon ligand binding that is anything like that predicted
for NDV HN.
To address the issue of the requirement for the minimal
interface form of HN for fusion, we took the approach of
precluding its formation by locking the protein into the exten-
sive interface form via the introduction of a pair of cysteine
mutations that form two disulfide bonds across the dimer in-
terface. As proof of principle, we first produced and charac-
terized a form of hPIV3 HN with monomers in each dimer
linked by a single intermonomeric disulfide bond and showed
that it retains fusion-promoting activity. An NDV HN protein
carrying T216C and D230C mutations also forms disulfide-
linked homodimers, which are formed both intracellularly and
in the absence of receptor binding and are efficiently expressed
at the cell surface. Most importantly, the mutated protein
promotes fusion more efficiently than the parental wild-type
(wt) protein. With the caveat that the disulfide-linked form of
the protein could be promoting fusion via a radically different
mechanism than the parent protein, these findings question
both the minimal interface structure for NDV HN and its role
MATERIALS AND METHODS
Cells. BHK-21F cells (provided by Rebecca Dutch) were maintained in Dul-
becco modified Eagle medium with high glucose, supplemented with 5% fetal
calf serum, 20 mM L-glutamine, 4 U of penicillin/ml, and 4 ?g of streptomycin/
ml. All tissue culture reagents were obtained from Invitrogen (Carlsbad, CA).
Recombinant plasmids and site-directed mutagenesis. The preparation of the
HN gene from the L-Kansas (L) strain of NDV in the pBluescript SK(?)
(Stratagene Cloning Systems, La Jolla, CA) was described previously (28), as
were the preparations of the HN and F genes from the Australia-Victoria (AV)
strain of NDV (8, 33) and the HN and F genes of hPIV3 (10).
Site-directed mutagenesis was performed as described previously (4). Briefly,
single-stranded DNA template was rescued by R408 helper phage (Stratagene)
in CJ236 cells. Mutagenesis primers (Integrated DNA Technologies, Coralville,
IA) were annealed to the template and extended with T4 DNA polymerase, and
the ends were ligated with T4 DNA ligase. The mutagenesis reactions were
transformed into DH5? cells that were then selected for ampicillin resistance.
Identification of colonies carrying mutated genes was facilitated by screening for
the presence of a unique restriction site introduced by each mutagenic primer.
Multiple clones were characterized for each mutated DNA, and the presence of
the desired mutation(s) was confirmed by DNA sequencing.
Transient-expression system. Wt and mutated HN proteins were expressed
using the vaccinia virus T7 RNA polymerase expression system (13), as described
previously using 1 ?g of each plasmid (5). All experiments were performed in
six-well plates seeded a day earlier at 4 ? 105cells per well.
Functional assays. Cell surface expression was quantitated by flow cytometric
analysis using a mixture of monoclonal antibodies (MAbs) for NDV HN, includ-
ing HN1b, HN2a, HN3c, HN4a, HN14f, and HN23a(17, 18, 23, 24), and a MAb
specific for hPIV3 HN (American Type Culture Collection, Manassas, VA).
Hemadsorption (HAd) activity was determined by the abilities of the expressed
HN proteins to adsorb guinea pig erythrocytes (Bio-Link, Inc., Liverpool, NY)
and quantitated as described previously (31). The NA activity of cell surface HN
was determined colorimetrically using sialyllactose (Sigma Chemical Co., St.
Louis, MO) as a substrate. For NDV HN, cell monolayers were incubated with
substrate for 20 min in 0.1 M sodium acetate (pH 6) as described previously (31).
To determine the NA activity of hPIV3 HN, the assay was modified such that cell
monolayers were incubated for 1 h at pH 4.8. For fusion pictures, transfected
monolayers were fixed with methanol and stained with Giemsa stain (Sigma).
The abilities of the mutated HN proteins to complement the homologous F
protein in the promotion of fusion were quantitated by using a content-mixing
assay, which measures ?-galactosidase activity in target cells after fusion induced
by HN-F-expressing effector cells (31).
Immunoprecipitation. The immunoprecipitation protocol has been described
previously (32). Briefly, at 20 to 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 ?Ci of Expre35S[35S] cysteine-methionine
labeling mix (Perkin-Elmer, Boston, MA) and chased for 90 min with growth
medium. The cells were lysed in lysis buffer (phosphate-buffered saline contain-
ing 1% Triton X-100, 0.5% deoxycholate, 30 mM N-ethylmaleimide, and 1 mM
phenylmethylsulfonylfluoride), and the NDV HN proteins were immunoprecipi-
tated either with a cocktail of MAbs or with individual MAbs. Immunoprecipi-
tation of hPIV3 HN was performed according to the same protocol except using
a polyclonal guinea pig antiserum prepared against the whole virus. 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) in the presence (10% gel) or absence (7.5%) of
The introduction of a disulfide bond across the dimer inter-
face of hPIV3 HN does not impair its fusion-promoting activ-
ity. X-ray crystallographic studies of the hPIV3 HN protein
indicate that its role in fusion requires only a minor change in
the structure of the dimer interface (27). Based on this, it is
reasonable to expect that the introduction of an intermono-
meric disulfide bond across the dimer interface of the protein
may not interfere with its ability to complement the homolo-
gous F protein in the promotion of fusion.
To test this hypothesis, we sought to identify residues di-
rectly apposed across the dimer interface at which cysteine
mutations could be introduced, which would have the potential
to form intermonomeric disulfide bonds. Figure 1A shows that
the S554 residues on the two monomers in the hPIV3 HN
dimer are very close across the dimer interface. In this posi-
tion, it seemed possible that the introduction of an S554C
mutation would result in the formation of an intermonomeric
VOL. 82, 2008ENGINEERED DISULFIDE BONDS IN NDV HN PROTEIN10387
disulfide bond. Thus, S554C-mutated hPIV3 HN was pre-
pared, and its oligomeric structure was evaluated by SDS-
PAGE under both nonreducing and reducing conditions (Fig.
1B). In the absence of BME, the wt L HN protein migrates as
a monomer of ?70 kDa. However, the S554C-mutated protein
migrates at a much slower rate as two distinct bands under
nonreducing conditions; there is minimal monomer present.
The slower-migrating form has an estimated molecular mass of
147 kDa, which is only slightly greater than that expected for a
dimer. The more diffuse band, estimated to be 128 kDa, is
smaller than expected for a dimer. In the presence of BME
(Fig. 1B), the S554C-mutated protein comigrates exclusively
with the wt monomer. This confirms that the monomers in
each dimer are disulfide linked. As a control, an S554A-mu-
tated protein migrates as a monomer in both gels.
The effect of the intermonomeric disulfide bond on the func-
tions of the mutated protein relative to those of wt hPIV3 HN
was determined. The disulfide-linked protein is efficiently ex-
pressed at the cell surface, as detected by flow cytometry
(90.5% ? 8.6% of wt) and exhibits greater-than-wt NA activity
(127.9% ? 19.9% of wt). The HAd activity of the disulfide-
linked molecule was determined at both 4 and 37°C. As shown
in Fig. 1C, the HAd activity of the wt protein is reduced
significantly at 37°C relative to that at 4°C. This is to be ex-
pected, given that NA is functional at the higher, but not the
lower, temperature. However, the disulfide-linked protein ex-
hibits a different profile. Its HAd activity is more than one-
third greater at 37°C than it is at the lower temperature, sug-
gesting that the disulfide bond may stabilize receptor binding
at the higher temperature. Finally, the fusion-promoting activ-
ity of the mutated hPIV3 HN protein was compared to that of
the wt protein. Quantitation of the extent of fusion of the
mutated protein in the content mixing assay reveals that it
promotes fusion 94.6% ? 13.1% as efficiently as the wt protein.
Thus, the presence of a disulfide bond across the dimer inter-
face of hPIV3 HN does not impair the ability of the protein to
complement the F protein in the promotion of fusion. This is
consistent with the prediction from the crystallographic data
that the role of hPIV3 HN in the promotion of membrane
fusion involves no change in its dimer interface.
Residues 216 and 230 are juxtaposed across the dimer in-
terface in the extensive interface form of NDV HN but far
apart in the minimal interface form. Interpretation of the
structural data for liganded and unliganded NDV HN has led
to the prediction that the protein undergoes a drastic confor-
mational change during fusion that converts it from a form
having a minimal dimer interface to one with a much more
extensive interface (6). To test this hypothesis, we reasoned
that the introduction of a disulfide bond in the appropriate
position across the dimer interface of the extensive interface
form would preclude formation of the minimal interface form
of the protein. If this disulfide bond impaired fusion, it would
argue in favor of the model. However, if an HN protein disul-
fide linked in this way were to retain significant fusion-promot-
ing activity, this would argue that the role of NDV HN in the
promotion of fusion does not require a drastic rearrangement
of the dimer interface.
Thus, as we did for hPIV3 HN, we looked for residues that
are in close apposition across the dimer interface in the exten-
sive interface conformation of NDV-L HN. We could find no
FIG. 1. hPIV3 HN S554 residues in the two monomers are in close
contact across the dimer interface and mediate an intermonomeric
disulfide bond, which does not impair fusion. (A) Top view of the
hPIV3 HN homodimer with the S554 residue on each monomer shown
in space-fill mode in blue on one monomer and yellow in the other.
The figure was generated with Swiss PDB-Viewer using the structure
of Lawrence et al. (27). (B) BHK cells were transfected with vector, wt
hPIV3 HN, or hPIV3 HN carrying either a S554C or S554A (as a
control) mutation, starved for cysteine and methionine for 1 h, labeled
for 3 h, and chased with complete medium for 90 min. The cells were
lysed in the presence of N-ethylmaleimide, the hPIV3 HN protein was
immunoprecipitated, and the samples were run either with or without
BME. The numbers in the lanes marked “M” indicate the migration
rates of markers in kilodaltons. (C) The HAd activities of wt and
S554C-mutated HN expressed at the surface of BHK cells were as-
sayed at 4 and 37°C. The error bars represent the standard deviations
of a minimum of five determinations.
10388MAHON ET AL. J. VIROL.
single residue that meets this requirement. However, residues
T216 and D230 from opposite monomers are in close contact
across the dimer interface in the extensive interface form (Fig.
2A). The introduction of cysteine mutations at these positions
has the potential to introduce two disulfide bonds at opposite
ends of the dimer interface. As shown in Fig. 2B, residues 216
and 230 are approximately 40 Å apart in the minimal interface
form of the protein, making it impossible for cysteines intro-
duced at these two positions to take part in a disulfide bond
while maintaining this structure.
T216C-D230C-mutated NDV-L HN is disulfide linked.
NDV-L HN carrying T216C and D230C mutations was pre-
pared by site-directed mutagenesis, and its oligomeric struc-
ture was analyzed by SDS-PAGE under nonreducing (Fig. 3A)
and reducing (Fig. 3B) conditions. wt L HN migrates as a
monomer (74 kDa) under both reducing and nonreducing con-
ditions. This is expected, as the protein does not possess inter-
monomeric disulfide bonds. As a control, the HN from the AV
strain of NDV migrates as a protein of 135 kDa in the absence
of BME, similar to the rate expected for a dimer. This is to be
expected, since its monomers are linked by an intermonomeric
disulfide bond mediated by a cysteine at position 123 in the
stalk region (40). The mutated form of L HN migrates at a
much slower rate under nonreducing conditions and signifi-
cantly slower than the AV HN dimer, predominantly at the
rate expected for a protein of 161 kDa. The 161-kDa protein is
slightly larger than expected for a dimer (148 kDa) but cer-
tainly not large enough to be either a trimer or tetramer. A
small amount of the protein migrates as a monomer and there
are also two minor bands migrating at 106 and 128 kDa. All of
the mutated L HN protein comigrates with the wt protein
under reducing conditions (Fig. 3B). These results confirm that
the monomers in the T216C-D230C-mutated L HN are linked
by intermonomeric disulfide bond(s). In addition, the presence
of N-ethylmaleimide in the lysis buffer eliminates the possibil-
ity that these bonds are formed only upon cell lysis.
The disulfide-linked form of NDV-L HN is efficiently ex-
pressed at the cell surface and promotes fusion more effi-
ciently than wt HN. The effect of the intermonomeric disulfide
bonds on the structure and function of the NDV-L HN protein
was determined. The T216C-D230C-mutated protein is effi-
ciently expressed, as detected by flow cytometry using a mix-
ture of HN-specific MAbs (118.0% ? 8.6% of the wt). How-
ever, flow cytometry does not distinguish between covalently
and noncovalently linked oligomers at the cell surface. To
confirm that the engineered, covalently linked dimers are, in-
deed, fully surface expressed, we immunoprecipitated HN
from the surface of cells expressing the mutated protein. Fig-
ure 4 shows that the disulfide-linked form of the mutated
protein can be immunoprecipitated from the surface of trans-
fected cells, confirming that the disulfide-linked form is effi-
ciently transported to the cell surface. Also, noteworthy is the
fact that the 106- and 128-kDa forms are not detectable at the
cell surface. This confirms that the 161-kDa form of the disul-
fide-linked mutated protein is by far the predominant one at
the cell surface.
To determine the effect of the intermonomeric disulfide
FIG. 2. NDV HN residues 216 and 230 from different monomers are closely apposed across the dimer interface in the extensive interface form
but far apart in the minimal interface form. Membrane views of the NDV HN dimer in the extensive interface form (A) and in the minimal
interface form (B), showing residues T216 and D230 from one monomer in blue and from the other monomer in yellow, are presented. The figures
were generated with Swiss PDB-Viewer, using the structures of Crennell et al. (6).
FIG. 3. T216C-D230C-mutated NDV-L HN is a disulfide-linked
dimer, which is expressed at the cell surface. BHK cells were trans-
fected with vector alone, AV HN, L HN, or T216C-D230C-mutated L
HN. At 22 h posttransfection, the monolayers were starved for cysteine
and methionine for 1 h, labeled for 3 h, and chased for 90 min. The
cells were lysed in the presence of N-ethylmaleimide, and the HN
protein was immunoprecipitated with a mixture of anti-HN-specific
MAbs. The samples were subjected to SDS-PAGE either without
(A) or with BME (B). The numbers in the lanes marked “M” indicate
the migration rates of markers in kilodaltons.
VOL. 82, 2008 ENGINEERED DISULFIDE BONDS IN NDV HN PROTEIN10389
bonds on the fusion-helper function of the protein, we com-
pared the abilities of wt and disulfide-linked HN to comple-
ment the NDV F protein in the promotion of fusion. As shown
in Fig. 5, the presence of the intermonomeric disulfide bonds
does not impair fusion (compare Fig. 5C to B). Indeed, quan-
titation of the extent of fusion in the content mixing assay
reveals a ca. 50% increase in fusion by the mutated protein
relative to the wt (Fig. 5D). Thus, prevention of formation of
the minimal interface form of HN clearly does not impair
fusion but rather appears to enhance it.
The intermonomeric disulfide bonds alter the receptor-
binding properties of NDV HN. To try to understand the basis
for the increased fusion promotion activity of the disulfide-
linked protein, its receptor-binding properties were compared
to those of the wt protein. This was done by comparing the
HAd activities of the two proteins at both 4 and 37°C. The wt
HN protein hemadsorbs significantly more efficiently in the
cold (optical density at 540 nm [OD540] of 0.133) than it does
at 37°C (OD540of 0.088) (Fig. 6A). The NA activity of HN is
not functional in the cold, while it acts to dissociate the HN-
receptor complex at 37°C. However, the disulfide-linked pro-
tein has the opposite phenotype, hemadsorbing more than
twofold better at 37°C (OD540of 0.172) than it does in the cold
(OD540of 0.075) (Fig. 5A). This is similar to the profile exhib-
ited by disulfide-linked hPIV3 HN (Fig. 1C). Indeed, the di-
sulfide-linked NDV-L HN hemadsorbs better at the elevated
temperature than does the wt protein in the cold. This could be
related to the sharp decrease in NA activity exhibited by the
FIG. 4. The disulfide-linked form of the T216C-D230C-mutated
NDV-L HN is expressed at the cell surface. Cells were transfected as
indicated and labeled for 3 h with 300 ?Ci of Expre35S[35S] cysteine-
methionine labeling mix. The cells were then placed on ice and washed
twice with ice-cold phosphate-buffered saline containing 0.1 mM CaCl2
and 1 mM MgCl2(PBS-CM). The monolayers were incubated for 1 h
in the cold with 2 ml of a mixture of anti-HN hybridoma supernatants,
washed five times with cold PBS-CM, and lysed, and HN was immu-
noprecipitated as described above, followed by SDS-PAGE without
BME. The numbers in the lane marked M indicate the migration rates
of markers in kilodaltons.
FIG. 5. T216C-D230C-mutated NDV-L HN promotes fusion more effectively than the wt protein. BHK cell monolayers were transfected with
vector alone (A), wt HN and F (B), or T216C-D230C-mutated L HN and wt F (C) and stained for fusion. Arrows indicate syncytia. (D) The
promotion of fusion by wt F complemented with either wt L HN or T216C-D230C-mutated L HN was quantitated in the content-mixing assay.
The error bars represent the standard deviations of a minimum of five determinations.
10390 MAHON ET AL.J. VIROL.
mutated protein (25.2% of wt NDV-L HN) but could also be
due, in part, to a stabilization of the second sialic acid binding
site at the membrane-distal end of the dimer interface.
The data shown in Fig. 6B illustrate an additional point. This
figure compares the HAd activity of wt NDV-AV HN at 4 and
37°C. As discussed previously, monomers of this HN protein
are linked by intermonomeric disulfide bonds between cys-
teines in the stalk region. This protein exhibits a profile similar
to that of wt L HN, i.e., slightly decreased HAd at 37°C relative
to that in the cold. Thus, HN carrying a naturally occurring
intermonomeric disulfide bond in its stalk does not exhibit the
same HAd phenotype as a protein with intermonomeric disul-
fide bonds in the globular domain. The altered HAd profile is
a function of the presence of two intermonomeric disulfide
bonds and/or their placement in the globular head.
Binding to receptors is not required for formation of the
intermonomeric disulfide bond in T216C-D230C-mutated
NDV HN. It was originally proposed that the conversion of
NDV HN from the minimal interface form to the extensive
interface conformation is triggered at the cell surface by its
binding to receptors (6). Assuming that the intermonomeric
disulfide bonds between residues 216 and 230 cannot exist in
the minimal interface form of the protein, this predicts that the
disulfide-linked protein will not be present prior to HN reach-
ing the cell surface and binding to receptors.
We took two approaches to test this hypothesis. First, we
looked for the presence of the disulfide-linked dimer inside the
cell. In the experiments shown in Fig. 3, designed to detect cell
surface HN, we expressed the mutated protein, radiolabeled
for 3 h and incubated in chase medium for 90 min. Here, in
order to determine whether the disulfide-linked, T216C-
D230C-mutated HN is present intracellularly, we labeled for
only 30 min and did not use a chase. Since the half time for
NDV HN to reach the surface has been determined to be 78
min (35), we should be dealing exclusively with intracellular
HN. As shown in Fig. 7A, the disulfide-linked dimer of the
mutated protein can be detected in significant amounts under
these conditions, indicating that the intermonomeric disulfide
bond forms before the protein has reached the surface. As a
control, the same can be said for the HN from the AV strain,
which is linked via a disulfide bond in the stalk (40). Again, the
presence of N-ethylmaleimide in the lysis buffer ensures that
the dimer is formed intracellularly. These results call into ques-
tion the idea that the extensive interface conformation of NDV
HN is formed at the cell surface triggered by the binding of HN
to cellular receptors. Note that the amounts of the 106- and
128-kDa forms of the mutated L HN protein are minimal
compared to the 161-kDa form.
As another approach to the question of the relationship
between formation of the extensive interface conformation and
receptor binding, we have taken advantage of a mutation pre-
viously identified in NDV HN that completely eliminates the
ability of the protein to mediate receptor binding. A D198R
mutation in the NA active site of NDV-AV HN results in a
totally a functional protein, i.e., one that lacks NA, attachment,
and fusion-promoting activities, despite efficient expression at
the cell surface (9). If HN does not assume the extensive
interface form until receptor binding, HN carrying a D198R
mutation should remain in the minimal interface form and
D198R-mutated HN carrying T216C and D230C mutations
should not be capable of forming the intermonomeric disulfide
To test this, we prepared D198R-mutated NDV-L HN both
with or without the two cysteine mutations and confirmed that
both proteins are expressed and lack appreciable HAd activity.
Indeed, no HAd activity at all can be detected for D198R-
mutated wt L HN at 4°C, and it exhibits only 5.5% ? 0.3% of
the activity of the wt protein at 37°C. No HAd activity can be
detected for the D198R-T216C-D230C-mutated protein at ei-
ther temperature. Similarly, the NA activities of the D198R-
mutated proteins are reduced to very minimal levels. D198R-
mutated L HN and D198R-T216C-D230C-mutated L HN
exhibit only 2.4% ? 2.0% and 1.5% ? 0.6% of the NA activity
of the wt protein, respectively.
The D198R-mutated proteins were expressed in BHK cells,
labeled, and chased to the surface. The cells were lysed in the
FIG. 6. The presence of the intermonomeric disulfide bonds in
NDV-L HN alters the receptor recognition profile of the protein.
(A) The ability of wt and T216C-D230C-mutated L HN expressed at
the surface of BHK cells to HAd guinea pig erythrocytes was assayed
at 4 and 37°C. (B) The HAd activity of wt HN from the AV strain was
compared at 4 and 37°C. The error bars represent standard deviations
of a minimum of five determinations.
FIG. 7. The intermonomeric disulfide bonds form in NDV-L HN
both intracellularly and in the absence of receptor binding. At 22 h
posttransfection, BHK cells expressing either vector alone or the HN
protein shown were starved for cysteine and methionine and labeled
for 30 min without a subsequent chase (A) or labeled for 3 h followed
by a 90-min chase (B). The cells were then lysed, HN was immuno-
precipitated using a mixture of HN-specific MAbs, and HN was dis-
played by SDS-PAGE in the absence of BME. The numbers in the lane
marked “M” indicate the migration rates of markers in kilodaltons.
VOL. 82, 2008 ENGINEERED DISULFIDE BONDS IN NDV HN PROTEIN10391
presence of N-ethylmaleimide, and HN was immunoprecipi-
tated and analyzed by SDS-PAGE (Fig. 7B). The presence of
the D198R mutation and the resulting loss, or near loss, of
receptor-binding activity have no discernible effect on the
amount of the protein that migrates as a disulfide-linked
dimer. Interestingly, the amounts of the minor bands that
migrate ahead of the dimer are decreased in the D198R-mu-
tated, disulfide-linked protein relative to the D198R-mutated
wt protein (compare lanes 4 and 5 in Fig. 7B). These results are
consistent with the conclusion that receptor binding is not
required for the formation of the intermolecular disulfide
bond, suggesting that it is also not required for the formation
of the extensive interface form of the protein. The relatively
more diffuse migration patterns for the D198R-mutated wt and
disulfide-linked proteins are consistent with our previous re-
sults (9) and are due to their lack of detectable NA activity,
which results in molecules with various levels of sialylation.
The presence of the intermonomeric disulfide bonds in-
creases the avidity with which MAbs to two antigenic sites
recognize NDV-L HN. We initially identified four antigenic
sites (sites 1 to 4) in the globular domain of the AV strain of
NDV HN (18). Subsequently, three additional sites, which
overlap two of the original ones in competition antibody bind-
ing and additive neutralization assays, were identified and
named site 12 (overlaps site 1 and 2), site 14 (overlaps sites 1
and 4), and site 23 (overlaps sites 2 and 3) (17, 20, 23).
Although we have demonstrated that a cocktail containing a
mixture of these MAbs efficiently recognizes T216C-D230C-
mutated NDV-L HN protein, we wanted to explore the possi-
bility that the presence of the intermonomeric disulfide bonds
might alter the antigenic structure of HN in a site-specific way.
Thus, lysates from cells expressing the HN from the AV strain
(against which the MAbs were made), the L strain, and the
disulfide-linked mutated form of L HN were individually im-
munoprecipitated with MAbs to six of the seven sites (Fig. 8A).
(MAbs to site 12 were not tested as they immunoprecipitate
HN only weakly.) The site 14 MAb is a positive control; it
recognizes a linear epitope (24) that is highly conserved in all
NDV strains tested (17). (All of the other MAbs recognize
conformational epitopes.) As expected, this MAb efficiently
immunoprecipitates all three proteins (Fig. 8A). The site 23
MAb serves as a negative control, as it is highly specific for the
AV strain due to a mutation at position 193 (23). As expected,
this MAb efficiently immunoprecipitates AV HN, but neither
wt nor mutated L HN (Fig. 8A). A similar result was obtained
with the site 2 MAb, which we have previously shown recog-
nizes L HN very weakly and apparently not with high enough
avidity to immunoprecipitate it (22).
However, interesting results were obtained with the MAbs
to the three remaining antigenic sites. Whereas the site 3 and
4 MAbs fail to immunoprecipitate the parental L HN protein,
they do immunoprecipitate the disulfide-linked mutated form
FIG. 8. The presence of the intermonomeric disulfide bonds in NDV-L HN renders the protein capable of being immunoprecipitated with
MAbs to antigenic sites 3 and 4. (A) BHK cells expressing wt AV HN, wt L HN, and the disulfide-linked mutated form of L HN were starved,
labeled, and chased as described in Fig. 3. The cells were then lysed and HN immunoprecipitated with individual MAbs to the antigenic sites shown,
and the immunoprecipitates were displayed on SDS-PAGE in the presence of BME. (B) BHK cells expressing vector alone, wt AV or L HN, or
L HN carrying the mutation(s) shown were treated as in panel A. HN was immunoprecipitated with MAb to either site 3 or 14, and the
immunoprecipitates were analyzed by SDS-PAGE in the presence of BME. (C) BHK cells expressing wt AV-HN, L HN, or T216C-D230C-mutated
L HN were immunoprecipitated with MAbs to site 3, 4, or 14, and the immunoprecipitates were displayed on SDS-PAGE in the absence of BME.
The numbers in the lane marked “M” indicate the migration rates of markers in kilodaltons.
10392 MAHON ET AL.J. VIROL.
of the protein quite efficiently. This is especially true of the site
3 MAb (Fig. 8A). Thus, the introduction of the disulfide bonds
in L HN renders the protein capable of being immunoprecipi-
tated by two MAbs (prepared against another strain of the
virus) that do not bring down the parental wt protein. In
addition, while the site 1 MAb immunoprecipitates L HN
weakly, it loses this ability with the mutated protein (Fig. 8A),
indicating that this site is also altered by the introduction of the
disulfide bonds, although apparently to a lesser extent.
As a negative control, we generated mutated forms of wt and
disulfide-linked L HN carrying a D287N mutation and ana-
lyzed them by immunoprecipitation and SDS-PAGE (Fig. 8B).
The D287N mutation was identified in a variant of the AV
strain that escapes neutralization by the site 3 MAb by the
addition of an N-glycan at this position (21). As a control, AV
HN, L HN and all its mutated forms are efficiently immuno-
precipitated by the site 14 MAb. The slower migration rate of
the wt L HN and T216C-D230C-mutated L HN proteins, car-
rying the additional D287N mutation, relative to the respective
parental wt proteins, confirms the presence of the supernumer-
ary N-glycan. As expected, neither of the D287N-mutated L
HN proteins was efficiently immunoprecipitated by the site 3
MAb (Fig. 8B). This confirms that the immunoprecipitation of
the disulfide-linked mutated protein by the MAb is specific.
Thus, linking the monomers in the L HN dimer by intermo-
lecular disulfide bonds alters the antigenic structure of the
protein in a site-specific way.
Having shown that the T216C and D230C mutations render
the L HN protein capable of being immunoprecipitated by
MAbs to sites 3 and 4, we wanted to confirm that it is actually
the dimer form of the protein that is immunoprecipitated by
these antibodies. To do so, we compared immunoprecipitates
obtained with MAbs to sites 3, 4, and 14 from cells expressing
the HN proteins from strain AV, L, and the double-cysteine-
mutated form of the latter on SDS-PAGE under nonreducing
conditions (Fig. 8C). The immunoprecipitates of the mutated
protein obtained with MAbs to both sites 3 and 4 exhibit a
band that comigrates with the dimer form present in the site 14
immunoprecipitate. This confirms that it is, indeed, the disul-
fide-linked dimer that is immunoprecipitated by the MAbs to
sites 3 and 4.
Attempts to disulfide link NDV-L HN dimers. The NDV HN
protein is a tetramer, composed of a pair of dimers. Although
our results with the mutated protein in which the two mono-
mers in each dimer are linked by intermonomeric disulfide
bonds argue against a drastic rearrangement of the dimer in-
terface during fusion, they do not address the possibility that
fusion involves a dissociation of the dimers in each tetramer.
We have tried to address this possibility by introducing disul-
fide bonds between dimers across the tetramer interface. As we
did for the dimer interface, we identified pairs of residues that
are closely apposed across the tetramer interface, mutated
them to cysteines, and looked for the formation of disulfide-
linked tetramers in SDS-PAGE. We made a total of three
doubly mutated NDV-L HN proteins: G134C-T232C, T216C-
K536C, and T255C-R480C. All three combinations have the
potential to form two disulfide bonds between each pair of
dimers in the tetramer. While the mutated proteins are ex-
pressed, hemadsorb, and promote fusion comparable to the wt
L HN protein (data not shown), disulfide-linked tetramer for-
mation was inefficient. Thus, we cannot say yet whether the
role of HN in fusion involves an alteration of the tetramer
X-ray crystallographic studies of unliganded and ligand-
bound forms of the HN proteins of hPIV3 (27) and PIV5 (47)
indicate that the dimer interfaces of both proteins undergo
only minor conformational changes after interaction with re-
ceptors. This is in stark contrast to the results from similar
studies of NDV HN, which led to the conclusion that, during
fusion, it undergoes a drastic conformational rearrangement
triggered by receptor binding that results in its conversion from
a form with minimal dimer interface contacts to one with a
much more extensive interface (6). Thus, these studies led to
the conclusion that the mechanism by which NDV HN con-
tributes fusion-helper function to its homologous F protein
may be quite different from those of the other two viruses.
However, the model for the role of the minimal interface
conformation of HN in fusion is confounded by the fact that, in
this structure, residue G124 from one monomer of the dimer is
62 Å away from the same residue on the other monomer. This
is difficult to reconcile with the fact that the two G124 residues,
which are at the top of the stalk, would be expected to be
closely aligned in the monomers in each dimer, especially in
strains in which cysteines at position 123 mediate an intermo-
nomeric disulfide bond between monomers in the same dimer
(40). This led even the original authors to question the phys-
iological significance of the minimal interface form (6).
We have prevented the formation of the minimal interface
form of the protein by the introduction of intermonomeric
disulfide bond(s) across the dimer interface in the globular
domain. The presence of the disulfide bond(s) does not impair
fusion, as would be expected if the minimal interface form of
the protein were required for fusion. These results argue very
strongly that neither the minimal interface form of NDV HN
nor a gross conformational change in the protein is required
for NDV-L HN to complement the F protein in the promotion
of fusion. However, we cannot rule out the possibility that the
HN protein may undergo a change within the constraints im-
posed by the intermonomeric disulfide bonds, i.e., similar to
those identified for hPIV3 HN. Indeed, such a change might be
instrumental to the formation of the second site at the dimer
interface. Furthermore, we also cannot rule out the possibility
that the disulfide-linked NDV-L HN protein may promote
fusion by a totally different mechanism than does the wt pro-
tein. However, this seems unlikely since the two ostensibly
different mechanisms do not require any changes in the com-
plementary fusion protein.
The disulfide-linked mutated form of L HN exhibits a slower
migration rate (161 kDa) relative to the AV HN dimer (135
kDa). This may be a reflection of its having an additional
intermonomeric disulfide bond relative to AV HN and/or the
fact that the two intermonomeric disulfide bonds in the mu-
tated L HN protein are located in the globular head, while the
single one in AV HN is located in the stalk. There are also two
minor bands present in the immunoprecipitate with estimated
molecular masses of 106 and 128 kDa. The latter migrates at a
rate similar to that of the AV HN dimer. We postulate that this
VOL. 82, 2008 ENGINEERED DISULFIDE BONDS IN NDV HN PROTEIN10393
band may be made up of L HN linked by only a single inter-
monomeric disulfide bond. Most importantly, neither of these
minor forms is detectable at the cell surface. Also, they are
immunoprecipitated by the site 14, but not by the site 3 and 4
MAbs (Fig. 8C). Since, of the three MAbs, only the site 14
antibody recognizes a linear epitope, this could mean that the
minor forms represent misfolded forms of the protein.
The model further contends that the minimal interface form
of NDV HN serves to hold F in its prefusion conformation
through contacts mediated by residues exposed in the minimal
interface form and later sequestered in the extensive interface
conformation with this conversion triggered by receptor bind-
ing (41). We tested this by examining the relationship between
receptor binding and formation of the extensive interface form
of HN. We showed that the disulfide-linked dimer forms both
intracellularly and in the absence of receptor-binding activity.
These results confirm that, in contrast to the predictions of the
model, the extensive interface form of HN is formed before the
protein binds receptors.
Our results are also inconsistent with a peptide-based study
which claimed that the interaction with F is mediated by a
domain defined by residues 124 to 152 at the stalk-globular
head interface in NDV HN and it is the shift from the minimal
interface conformation to the extensive interface form that
leads to release of HN from a preformed complex with F and,
in turn, fusion (15). This is not surprising in light of our finding
that the introduction of a N-glycan within this domain does not
significantly reduce the level of fusion (32), and the over-
whelming body of evidence indicates that the F-interactive site
in NDV HN resides in its stalk region (10, 31, 32, 46). Taking
all this into consideration, it appears that, if HN does control
fusion by maintaining F in its prefusion state, this interaction is
most likely mediated by residues in the stalk.
The presence of the disulfide bonds across the dimer inter-
face of L HN not only enhances its fusion-promoting activity
but also leads to a remarkable shift in its receptor-binding
profile. While the mutations reduce the ability of the protein to
bind receptors in the cold, they significantly enhance it at 37°C.
Indeed, enhanced fusion may be causally related to increases
in receptor binding at 37°C. Our interpretation of this is that
the disulfide bonds stabilize the second sialic acid binding site
at the dimer interface. We postulate that this is the obverse of
our demonstration that the elimination of hydrogen bonds
across the dimer interface decreases binding at 37°C, in turn
sharply decreasing fusion (5). In retrospect, this destabilization
of the dimer interface may disrupt the second sialic acid bind-
ing site, although it cannot be ruled out that an intact dimer
interface is required for NDV HN?s role in fusion.
Disulfide-linked hPIV3 HN exhibits a receptor-binding pro-
file similar to that of the disulfide-linked NDV-L HN mutated
protein. Its HAd activity is greater at 37°C than it is at 4°C.
Although not identified in the crystal structure of the protein
(27), it has been proposed that hPIV3 HN, like NDV HN, also
possesses a second sialic acid binding at its dimer interface
(38). The slight increase in NA activity in the mutated hPIV3
protein supports the idea that its increased HAd at the higher
temperature is not related to changes in NA. Thus, the in-
creased HAd at 37°C relative to 4°C exhibited by the disulfide-
linked hPIV3 HN protein could be due to stabilization of a
second sialic acid binding site, if one exists in this protein.
Conclusive proof of the existence of a second site on hPIV3
HN awaits the detection of ligand bound to it.
Although the introduction of the intermonomeric disulfide
bonds in NDV-L HN does not grossly alter the conformation
of the protein, the immunoprecipitation assays with individual
MAbs indicate that it does alter the structure of some antigenic
sites. Most important is the demonstration that linking the
globular heads by intermonomeric disulfide bonds makes it
possible for MAbs to sites 3 and 4 to immunoprecipitate the
dimer form of the L HN mutated protein, whereas the parental
protein is not immunoprecipitated in appreciable amounts by
these MAbs. The failure of the site 3 MAb to immunoprecipi-
tate wt L HN is consistent with our previous observation that
this MAb binds NDV-L HN at very low avidity, as evidenced by
its failure to neutralize the NDV-L virus unless rabbit anti-
mouse immunoglobulin is added (17, 22). Thus, the intermo-
nomeric disulfide bonds apparently alter the globular domain
such that the avidity by which it is recognized by site 3 and 4
MAbs is increased sufficiently to make immunoprecipitation of
the protein possible.
Whereas MAbs to sites 1, 2, 12, 14, and 23 inhibit both
attachment and fusion (20), MAbs to sites 3 and 4 exhibit
unique functional inhibition profiles. Although they do not
inhibit viral attachment (20), both MAbs efficiently block fu-
sion-from-without (FFWO), a model for virus entry, while
MAbs to site 3, but not those to site 4, block fusion-from-within
(FFWI) (21). Thus, MAbs to these sites are capable of block-
ing virus-cell and/or cell-cell fusion at a step subsequent to
receptor binding. The acquisition of the ability to immunopre-
cipitate the disulfide-linked form of L HN suggests that site 3
and 4 MAbs recognize this form of the protein with increased
Amino acid residues in the globular domain of HN that
contribute to each of the seven sites have been identified by the
isolation and sequencing of antigenic variants (21, 23, 24). The
antigenic structure of the NDV HN globular domain generated
in this way is shown in Fig. 9. The inhibition of attachment by
MAbs to sites 1, 2, 12, 14, and 23 is consistent with their
localization either overlapping the NA active site (site 23), in
close proximity to the second sialic acid binding site (sites 2
and 12) or on the top surface of the globular domain (sites 1
and 14). However, both site 3 and site 4 are located on the
lateral surface of the globular head, which is quite a distance
from the receptor binding sites; this likely accounts for their
inability to inhibit attachment.
Thus, the introduction of disulfide bonds across the dimer
interface increases the avidity with which two MAbs, which
bind to the lateral surface of the globular domain and specif-
ically block fusion, interact with the HN from a heterotypic
strain of NDV. These results raise the possibility that disulfide-
linked L HN represents a form of the protein more relevant to
fusion and argues for a role for the lateral surface of the
globular domain of HN in fusion promotion, as proposed by
Yuan et al. (47), although not necessarily in mediating the
interaction with F. Alternatively, one could envision how the
binding of an antibody to the lateral surface of the globular
domain could sterically block the HN-F interaction, even if it is
mediated by the stalk of HN. Thus, as stated before, although
our data are consistent with the minimal interface form being
an artifact, they do not rule out the possibility that NDV-L HN
10394 MAHON ET AL.J. VIROL.
may undergo a much less drastic conformational change during
fusion from a form that is not recognized by MAbs to sites 3
and 4 to one that is recognized and is similar to the disulfide-
linked protein we have created by mutagenesis.
We thank Rebecca Dutch for the BHK-21F cells, Robert Lamb for
the NDV-AV F gene, Trudy Morrison for the NDV-AV HN gene,
Mark Galinski for the hPIV3 HN and F genes, and Bernard Moss for
the recombinant vaccinia virus. We also thank Theodore Jardetzky for
the coordinates for the NDV HN tetramer.
This study was supported by grant AI-49268 from the National
Institutes of Health.
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