Fusion activation by a headless parainfluenza virus
5 hemagglutinin-neuraminidase stalk suggests
a modular mechanism for triggering
Sayantan Bosea, Aarohi Zokarkara, Brett D. Welcha,b, George P. Lesera,b, Theodore S. Jardetzkyc,
and Robert A. Lamba,b,1
aDepartment of Molecular Biosciences andbHoward Hughes Medical Institute, Northwestern University, Evanston, IL 60208; andcDepartment of Structural
Biology, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Robert A. Lamb, August 8, 2012 (sent for review July 3, 2012)
The Paramyxoviridae family of enveloped viruses enters cells
through the concerted action of two viral glycoproteins. The
receptor-binding protein, hemagglutinin-neuraminidase (HN), H,
or G, binds its cellular receptor and activates the fusion protein, F,
which, through an extensive refolding event, brings viral and cel-
lular membranes together, mediating virus–cell fusion. However,
the underlying mechanism of F activation on receptor engagement
remains unclear. Current hypotheses propose conformational
changes in HN, H, or G propagating from the receptor-binding site
in the HN, H, or G globular head to the F-interacting stalk region.
We provide evidence that the receptor-binding globular head do-
main of the paramyxovirus parainfluenza virus 5 HN protein is
entirely dispensable for F activation. Considering together the
crystal structures of HN from different paramyxoviruses, varying
energy requirements for fusion activation, F activation involving
the parainfluenza virus 5 HN stalk domain, and properties of a chi-
meric paramyxovirus HN protein, we propose a simple model for
the activation of paramyxovirus fusion.
fusion triggering|hemagglutinin-neuraminidase structure|
protein refolding|viral membrane fusion
parainfluenza viruses (PIV) 1–5, mumps virus, Newcastle disease
virus (NDV), Sendai virus, measles virus, canine distemper virus
(CDV), Hendra virus, Nipah virus, respiratory syncytial virus,
and metapneumoviruses. The Paramyxoviridae are enveloped,
negative-stranded RNA viruses that enter cells by fusing their
envelope at neutral pH with the plasma membrane of the target
cell, releasing the viral genome into the cytoplasm in the form of
a ribonucleoprotein complex.
Paramyxovirus-mediated fusion depends on the concerted
actions of two glycoproteins, an attachment protein [hemaggluti-
nin-neuraminidase (HN), H, or G] and its cognate fusion (F)
protein, the latter initially folding into a metastable form. The at-
tachment protein is thought to trigger the fusion protein in a re-
ceptor-dependent manner (1–5). This triggering of the metastable
F (6) by the receptor-binding protein couples receptor binding of
refolds into a highly stable postfusion form (7). In the process, F
undergoes a series of structural rearrangements involving several
intermediates and brings about membrane merger (8).
HN proteins bind sialic acid as their receptor and also have
neuraminidase (receptor-destroying) activity. The PIV5 HN pro-
tein comprises 565 residues and has a short N-terminal cytoplas-
mic tail followed by a single transmembrane domain and a large
ectodomain (residues 37–565). The protein consists of a stalk re-
gion (residues 1–117) that supports a large globular head domain
(residues 118–565) containing the receptor-binding and neur-
aminidase-active site. X-ray crystal structures of the globular head
domain of PIV5, NDV, Nipah virus, Hendra virus, measles virus,
and human parainfluenza virus 3 (hPIV3) attachment proteins
he Paramyxoviridae include some of the great and ubiquitous
disease-causing viruses of humans and animals and include
have been obtained (9–16). The PIV5 HN globular head struc-
ture (16) reveals a neuraminidase-like fold with a six-bladed
β-propeller structure that is a common feature of the other
paramyxovirus HN/H/G head domains, regardless of receptor
specificity. The sialic acid-binding site is placed centrally within
the β-propeller. PIV5 HN exists as a pair of disulfide-linked
dimers with the disulfide bond at cysteine 111 (16, 17). These
dimers are associated noncovalently to form a dimer-of-dimer
oligomer (16, 18). The PIV5 HN dimer-of-dimer structure showed
the dimers arranged at an approximately 90° angle to each other
in an overall twofold symmetry creating a 657-Å2dimer-of-dimer
interface (16). This form of the tetrameric HN protein is referred
henceforth to as the “four-heads-up” form. The recently obtained
structure of the NDV HN globular head dimer also contains part
of the stalk revealing a four-helix bundle (4HB) (15). Despite a
strong structural similarity with the PIV5 HN globular head dimer
(16), NDV HN showed a different arrangement of the heads in
the tetramer, which we refer to hereafter as the “four-heads-
down” form. In this NDV HN structure, an interaction interface
was observed between residues of the globular head of NDV HN
and residues 83–114 in the NDV HN 4HB stalk domain. The
ability of the HN/H/G heads to adopt different interactions with
respect to the stalk is supported by electron micrographs of
purified PIV5 HN protein that indicate a variety of arrangements
of the heads (18). Recent biochemical evidence indicates sig-
nificant movement of the measles virus H protein head domains
that contributes to the ability of H to trigger F (19, 20). These
data suggest potential functional roles for the different confor-
mational arrangements observed in the crystal structures of the
paramyxovirus receptor-binding protein. In addition to the sim-
ilarities in their HN globular heads, the stalk regions of NDV
and PIV5 (15, 21) show a high degree of structural similarity
and overlapping regions of putative F interaction (21). Recent
studies have shown that sequences of other paramyxovirus re-
ceptor-binding protein stalks can be modeled as 4HBs based
on the crystal structures of the PIV5 HN and NDV HN stalk
Considerable evidence indicates that the stalk domains of
paramyxovirus HN/H/G proteins have a significant role in direct
interactions with the F protein (21–23, 25–34). In a number of
these studies, mutations in the stalk domains of paramyxovirus
receptor-binding proteins have been shown to block F activation,
presumably by blocking F–HN interactions.
Author contributions: S.B., A.Z., B.D.W., G.P.L., T.S.J., and R.A.L. designed research; S.B., A.Z.,
B.D.W., and G.P.L. performed research; S.B., A.Z., B.D.W., G.P.L., T.S.J., and R.A.L. analyzed
data; and S.B., T.S.J., and R.A.L. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1213813109PNAS Early Edition
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Assuming that interactions of HN/H/G with F probably occur
via the stalk region of the receptor-binding protein and that
concomitant alterations in this interaction may occur upon re-
ceptor binding to trigger F, it is possible that receptor binding by
the HN/H/G head domains induces conformational changes that
propagate down through the stalk on receptor binding (24, 35).
Here we provide evidence for a different hypothesis. We show
that residues 1–117 of the PIV5 HN stalk domain are sufficient
to activate F for fusion and that the HN globular head domain is
completely dispensable for PIV5 F triggering. We provide evi-
dence suggesting that the globular head domains of para-
myxovirus HN proteins play a regulatory role in fusion, possibly
via the movement of the heads; and that this role is maintained
even for a chimeric molecule possessing globular head and stalk
domains from different paramyxoviruses. Also, we show by using
previously characterized N-glycosylation mutants that the inter-
actions of the headless PIV5 HN 1–117 protein with F are similar
to the F–HN interactions of WT PIV5. To provide further evi-
dence for the regulatory nature of the globular head, we high-
light the roles of two important interfaces in the PIV5 HN
globular head domains. By using mutants located in the dimer-
of-dimer interface residues of the four-heads-up form (16) and in
the putative head–stalk interaction residues of PIV5 HN, mod-
eled on the four-heads-down NDV HN structure (15), we
demonstrate an effect on fusion activation on the disruption of
these interface interactions. We propose a simple model of
paramyxovirus fusion activation in which the receptor-binding
protein stalk domain provides the primary trigger in fusion and
the globular head domains undergo structural changes to regu-
late the activation of fusion.
PIV5 HN Stalk Domain 1–117 Is Sufficient to Activate Its Fusion Protein
and to Mediate Cell-to-Cell Fusion. Recently we determined the
structure of the PIV5 HN stalk domain from a construct ex-
pressed in insect cells containing stalk residues 56–117 (Fig. 1A)
crystal structures of the tetrameric PIV5 HN globular head domains (16) and the 4HB of the PIV5 HN stalk domain (21). Regions for which there is no known
structure (amino acids 1–58 and 103–117) are represented by dotted lines. CT, cytoplasmic tail; NA1–NA4, globular neuraminidase head domains. (B) Sche-
matic representation of the PIV5 HN full-length WT protein (amino acids 1–565) (Upper) and the PIV5 HN stalk protein (amino acids 1–117) lacking the
globular head domain (amino acids 118–565) (Lower). CT, cytoplasmic tail; TM, transmembrane domain. (C) Representative micrographs of syncytia showing
cell–cell fusion in BHK-21 cells transfected with F and WT HN or HN 1–117 stalk 18 h posttransfection. (D) Luciferase reporter assay of cell–cell fusion. (E and F)
Radioimmunoprecipitation of [35S]-TranS–labeled transfected 293T cell lysates, using PIV5 HN pAb R471 or a mixture of mAbs to the PIV5 HN globular head.
(E) WT HN protein analyzed on a 10% (wt/vol) SDS/PAGE gel. (F) HN 1–117 stalk protein analyzed on a 15% (wt/vol) SDS/PAGE gel. Numbers to the left of each
panel are molecular masses in kDa. (G) Representative graphs showing the detection of proteins at the cell surface of 293T cells transfected with WT HN or HN
1–117 stalk using flow cytometry. Surface proteins were detected using a mixture of HN mAbs or HN pAb R471. Average mean fluorescence intensities (MFI)
across three independent experiments are shown.
The PIV5 HN stalk domain is sufficient to activate F and mediate cell–cell fusion. (A) Structural view of the PIV5 HN full-length protein based on X-ray
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(21). This portion of the stalk is a 4HB that consists of a distinct
hydrophobic core. In light of recent data indicating that para-
myxovirus HN stalks have an essential role in fusion (21–23, 25,
36), we tested the function of the PIV5 HN stalk in a biological
context by removing the globular head domain (Fig. 1B). We used
an expression construct (PIV5 HN 1–117 stalk) lacking the entire
HN head (residues 118–565). However, the HN stalk construct
includes the intradimer disulfide bond at HN cysteine residue 111.
When the PIV5 HN 1–117 stalk construct was coexpressed
with PIV5 F in BHK-21 cells, significant cell–cell fusion char-
acterized by large syncytia, similar to the syncytia observed in
cells expressing WT F and HN, was observed (Fig. 1C). Quan-
tification of this fusion using a luciferase assay indicated ∼70–
75% fusion activity compared with WT F + WT HN (Fig. 1D).
The ability of the PIV5 HN 1–117 stalk to activate PIV5 F is
specific, because other F proteins from NDV HN, hPIV3 HN,
and hPIV2 HN could not be activated by the PIV5 HN 1–117
stalk. To detect the expression of HN 1–117 stalk protein in
mammalian cells, [35S] metabolically labeled proteins were
immunoprecipitated from transfected cell lysates using a poly-
clonal HN antibody, R471 (21). Additionally, as a control, a
mixture of PIV5 HN monoclonal antibodies (mAb 1b and mAb
4b) that are known to bind the globular head of PIV5 HN spe-
cifically were used (37). As expected, the PIV5 HN full-length
protein could be detected by the two mAbs and by the polyclonal
antibody (pAb) R471 (Fig. 1E), whereas the HN 1–117 stalk,
lacking the globular head domains could be detected using only
pAb R471 (Fig. 1F). A similar result was obtained when both the
expressed WT PIV5 HN and PIV5 HN 1–117 stalk proteins were
detected on the cell surface using pAb R471 and flow cytometry,
but only the full-length PIV5 HN protein could be detected by
the two mAbs (Fig. 1G). Collectively, these data indicate that
fusion activation of the PIV5 F protein can be mediated by ex-
pression of the HN 1–117 stalk domain alone. A factor that
could affect the level of fusion is the relative molar ratio of WT
HN to HN 1–117 stalk accumulating in cells, but this effect
cannot be determined readily because of the differential anti-
body reactivity to WT HN and the HN 1–117 stalk domain.
Absence of the PIV5 HN Globular Head Domain Energetically Favors
Fusion Activation. To explore further the mechanism by which the
HN 1–117 stalk domain activates fusion in the absence of the
head domain, the level of fusion activation by the PIV5 HN 1–
117 stalk protein and the WT HN protein at a suboptimal tem-
perature was compared. In a quantitative luciferase fusion assay
performed at 33 °C, fusion activation by WT HN was reduced
significantly compared with fusion at 37 °C. However, there was
very little reduction in the amount of fusion mediated by the
PIV5 HN 1–117 stalk at this suboptimal temperature (Fig. 2A).
These data suggest an energy requirement of WT PIV5 HN for
F activation that is observed only at a suboptimal temperature
and that this energy is required only when the head domains
PIV5 F-P22L is a hypofusogenic mutant of PIV5 F that needs
to overcome an increased activation barrier to transition into the
more stable postfusion form (38). PIV5 F-P22L is activated very
poorly by WT PIV5 HN at 37 °C (38). However, when F-P22L
was cotransfected with the PIV5 HN 1–117 stalk, a significant
amount of fusion (∼50% of WT HN), compared with minimal F-
P22L activation by the PIV5 HN full-length protein, was ob-
served in a quantitative fusion assay (Fig. 2B).
To examine the comparative populations of the prefusion
and the postfusion conformations of F or F-P22L at the cell
surface on activation by the PIV5 WT HN or the HN 1–117 stalk
protein, flow cytometry was performed using a F prefusion
conformation-specific mAb (F1a) (Fig. 2C) or a F postfusion
conformation-specific mAb 6-7 (Fig. 2D). The comparable
amounts of prefusion F detected on the surface of transfected
cells were in the order F-P22L + WT HN > WT F + WT HN >
WT F + HN 1–117 stalk (Fig. 2C). On the other hand, the
amounts of postfusion F detected on the cell surface were in the
reverse order compared with the amounts of prefusion F: WT
F + HN 1–117 stalk > WT F + WT HN > F-22L + WT HN
fusion activation. (A) Fusion activation at 33 °C (white bars) and 37 °C (gray
bars) by the PIV5 HN WT and HN 1–117 stalk protein. Fusion was measured
by a luciferase assay. (B) Luciferase reporter assay showing relative activation
of a hypofusogenic mutant of PIV5 F (F-P22L) by WT PIV5 HN protein in
comparison with activation by the PIV5 HN 1–117 stalk protein. (C and D)
Detection of pre- and postfusion PIV5 F and PIV5 F P22L proteins on the
surface of cells cotransfected with WT PIV5 HN or the PIV5 HN 1–117 stalk,
using flow cytometry. The amounts of surface antigen detected by an F-
prefusion conformation–specific mAb, F1a, (C) and an F-postfusion confor-
mation–specific mAb, 6-7, (D) are shown as a percentage MFI of the WT F +
WT HN sample (n = 3). (E) Representative electron micrographs of purified
PIV5 HN ectodomain showing different conformations of the dimer-of-di-
mer globular heads with respect to the stalk domain. (Upper) Conformations
of HN in which the stalk is exposed (four-heads-up–like arrangements).
(Lower) Examples of HN with the heads juxtaposed with the stalk domain
(four-heads-down–like arrangements). (Scale bar, 20 nm.)
Absence of the PIV5 HN globular head domain energetically favors
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(Fig. 2D). Taken together the data indicate that loss of the HN
head domain to generate the HN stalk 1–117 leads to an in-
creased ability to convert prefusion F to the postfusion form.
Furthermore, the data shown in Fig. 2 A and B indicate an en-
ergy requirement for HN for fusion activation, possibly to move
the head domains with respect to the stalk. Multiple arrange-
ments of the head domains with respect to the stalk domains are
possible, as seen in electron micrographs of purified PIV5 HN
ectodomain protein (Fig. 2E).
N-Linked Carbohydrate Chains in the Putative F-Interacting Region of
the PIV5 HN 1–117 Stalk Block Fusion Activation. Previously we de-
scribed PIV5 HN mutants that introduced sites for the addition
of N-linked carbohydrate chains into the HN stalk; these mutants
were designed to block F and HN interactions physically and
were based on a set of functional assays in which we defined
a putative region of F interaction on the PIV5 HN stalk (21). All
the PIV5 HN mutants showed a defect in fusion except one
(N102) in which an N-linked glycan chain was added to residue
102. Using the information gained from the HN mutants (21), we
introduced mutations in the HN 1–117 stalk to add glycosylation
sites at residues 60, 66, 67, 77, and 102 that mapped to the outer
surface of the 4HB (Fig. 3A). The HN 1–117 stalk N-glycosyla-
tion mutants were expressed in cells and were metabolically la-
beled, and the proteins were immunoprecipitated using HN pAb
R471 (Fig. 3B). The decrease in electrophoretic mobility of these
mutants compared with HN 1–117 stalk on a SDS/PAGE gel
indicates that these mutants are glycosylated. The minor HN-
specific bands possibly are the result of partial glycosylation at
a natural glycosylation site at N110. Glycosylation of these
mutants was confirmed by analyzing mutant HN proteins that
had been metabolically labeled in the presence of the N-linked
glycosylation inhibitor tunicamycin (5 μg/mL). All mutant pro-
teins showed faster electrophoretic mobility, as expected for
unglycosylated proteins. All HN 1–117 stalk N-glycosylation
mutants except N60 had relatively robust levels of protein
expression on the surface of transfected cells, ranging between
75–90% of that of the HN 1–117 stalk (Fig. 3C). The aberrant
migration of HN 1–117 N60 protein may be caused by altered
To demonstrate the specificity of the HN 1–117 stalk domain
in activating fusion, the HN 1–117 stalk N-glycosylation mutants
described above were coexpressed with F, and a quantitative
fusion assay was performed. As shown in Fig. 3D, mutants N60,
N66, N67, and N77 abolished fusion completely, whereas mutant
N102 had a low but significant level of fusion reflecting the
results obtained using full-length mutant HN proteins (21).
These data suggest that the stalk interacts with the F protein in
a similar manner, regardless of the presence or absence of the
HN globular head.
F Activation Mediated by the PIV5 HN 1–117 Stalk Does Not Depend
on Engagement of the Sialic Acid Receptor. For PIV5 WT HN the
globular head domains bind sialic acid, and this binding generally
is believed to act as the signal for F activation. To understand
fusion activation by the headless HN 1–117 stalk, we first elim-
inated the possibility that HN 1–117 stalk binds sialic acid. PIV5
HN 1–117 stalk showed no specific retention of chicken RBCs,
indicating that it had no specific sialic acid-binding capability
Unlike most paramyxoviruses, for PIV5 (isolate W3A) ex-
pression of F alone can mediate some fusion of transfected cells
in the absence of HN; however, coexpression of F and HN
greatly increases the extent of fusion (2). Additionally, we have
shown previously that heat can be used as a surrogate for HN in
triggering fusion (39). As shown in Fig. 4B, cells expressing F
alone that were incubated at 37 °C produced small syncytia, but
cells expressing F alone that were incubated at 42 °C produced
PIV5 HN 1–117 block fusion activation. (A) Schematic representation of posi-
linked carbohydrate chains were added by mutagenesis. The mutants are
named according to the residue carrying the N-glycosylation. (B) Migration
pattern of N-glycosylation mutants in the PIV5 HN 1–117 stalk on a 15% (wt/
vol) SDS PAGE gel. Polypeptides were immunoprecipitated from radiolabeled
lysates of transfected 293T cells using PIV5 HN pAb R471. Numbers on the left
are molecular masses in kDa. (C) Cell-surface expression of PIV5 HN 1–117 N-
glycosylation mutants determined using HN pAb R471 as measured by flow
cytometry (n = 3). (D) Fusion activation of PIV5 F by the HN 1–117 stalk and its
N-glycosylation mutants. Fusion was quantified using a luciferase reporter
assay as described in Experimental Procedures.
N-linked carbohydrate chains in the putative F-interacting region of
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syncytia that were comparable in size to those produced by cells
coexpressing F and HN that were incubated at 37 °C. Treatment
of cells with increasing concentrations of exogenous Clostridium
perfringens neuraminidase (0.025–0.075 U/mL), as expected,
blocked extensive syncytia formation in cells coexpressing F and
HN that were incubated at 37 °C (Fig. 4B). However, neuramini-
dase treatment of cells expressing F alone that were incubated at
37 °C or 42 °C had no effect on syncytia formation. At 37 °C the
formation of syncytia in cells coexpressing PIV5 HN 1–117 stalk
and F was unaffected by neuraminidase treatment, contrary to
the results obtained for WT F + WT HN expression.
To confirm that fusion activation did not require sialic acid
binding to the specific receptor-binding sites on the HN head
domains, cells expressing F alone, F and HN, or F and HN 1–117
stalk were treated with 0–10 mM 4-guanadino-2-deoxy-2,3-
didehydro-N-acetylneuraminic acid (zanamivir), the specific in-
hibitor of the HN catalytic site. In cells coexpressing F and HN
incubated at 37 °C, 5 mM zanamivir inhibited syncytia formation
(Fig. 4C). In contrast, in cells expressing either F alone incubated
at 42 °C or F and HN 1–117 stalk incubated at 37 °C, 10 mM
zanamivir did not inhibit syncytia formation (Fig. 4C). Taken
together, these data show that the promotion of fusion by the
HN 1–117 stalk (or by F alone when incubated at 42 °C) does not
depend on the engagement of the sialic acid receptor. These data
do not address whether PIV5 F has a cellular receptor molecule
or whether cell–cell contact with F expression is sufficient to
allow the formation of syncytia on the expression of HN 1–117
stalk or when F is heated to 42 °C.
Domain-Specific Functions of the HN Stalk and Energy Requirement
by the Globular Head During F Activation by a Chimeric Molecule. The
facts that the PIV5 HN 1–117 stalk alone can activate F and that
the PIV5 HN globular heads require energy for F activation at
33 °C led us to investigate if the head and the stalk of para-
myxovirus HN proteins could function as independent domains
linked through a flexible linker. To test this notion, we created
a chimeric HN protein by fusing the PIV5 HN 1–117 stalk to the
NDV HN protein head (residues 124–571) (Fig. 5A). The chi-
meric protein was expressed in cells and was detected at the cell
surface by flow cytometry using a mixture of NDV HN mono-
clonal antibodies (40). The PIV5-NDV HN chimera was ex-
pressed at the cell surface at ∼50% of WT NDV HN levels (Fig.
5B). Receptor binding by the NDV HN head in the chimeric
protein, as measured by a hemadsorption assay using chicken
RBCs, was 80% of WT NDV HN (Fig. 5C). The neuraminidase
activity of the PIV5-NDV HN chimera was similar to that of the
NDV HN protein when the differences in cell-surface expression
are taken into account (Fig. 5D).
When tested for its ability to activate F protein as measured by
a luciferase reporter assay, PIV5-NDV HN showed significant
amounts of fusion with PIV5 F. NDV F was not activated by
PIV5-NDV HN, showing that the specificity of the F protein
resides in the stalk (Fig. 5E and Fig. S1A). Because the PIV5 HN
1–117 stalk domain attached to a noncognate globular head
could activate F successfully, we investigated whether the PIV5-
NDV HN chimera would require energy to activate F at 33 °C, as
was observed in the full-length PIV5 HN protein (Fig. 2A). Fu-
sion quantified for PIV5 HN and PIV5-NDV HN at 33 °C
showed that the energy requirement of the PIV5-NDV HN
chimera was similar to that of the PIV5 HN protein (Fig. 5E),
unlike the PIV5 1–117 HN stalk, which was unaffected by tem-
perature (Fig. 2A). Additionally, like PIV5 HN-induced fusion
(Fig. 4B), fusion induced by the PIV5-NDV HN chimera could
be blocked by 0.1 U/mL C. perfringens neuraminidase (Fig. S1B).
HN 1–117 stalk. Chicken RBCs bound onto surfaces of transfected 293T cells were quantified by lysing the bound RBCs after extensive PBS washes and
measuring absorbance of chicken hemoglobin at 410 nm. (B and C) BHK-21 cells transfected with PIV5 F only or cotransfected with PIV5 F and PIV5 HN or PIV5
HN 1–117 stalk were incubated with C. perfringens neuraminidase or zanamivir at 37 °C or 42 °C. Cells were fixed, stained, and imaged 18 h posttransfection.
(B and C) Fusion activation in the presence of 0 U/mL, 0.025 U/mL, 0.05 U/mL, and 0.075 U/mL C. perfringens neuraminidase to remove most sialic acid linkages
(B) and with the addition of 0 mM, 0.25 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM zanamivir to inhibit PIV5 HN catalytic sites specifically (C).
F activation mediated by the PIV5 HN 1–117 stalk does not depend on engagement of the sialic acid receptor. (A) Receptor-binding activity of the PIV5
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Structurally Conserved Loops in the Paramyxovirus HN Globular Head
Domains Have Important Regulatory Roles in Fusion and Receptor
Binding. The two known positions of the HN head domains cre-
ate different surfaces for protein–protein interactions and make
unambiguous mutagenesis studies of function more challenging.
The four-heads-up position in the PIV5 HN atomic structure
(16) creates a dimer-of-dimer interface that buries a surface area
of 657 Å2(Fig. 6A). The four-heads-down conformation of the
NDV HN atomic structure (15) revealed an interface of in-
teraction between two globular NA head domains and the 4HB
of the stalk, burying a significant area (928 Å2) of protein–pro-
tein interaction. We aligned the structures of PIV5 HN head
domain (16) with the NDV HN head domain (15), highlighting
a region conserved structurally in the NA domains of both
viruses and that form a part of the stalk–head interface in NDV
HN (Fig. 6B). Side chains of residues P243, P130, and G134 in
NDV HN and their corresponding residues P233, N121, and
N125 in PIV5 HN are in positions that potentially form contacts
between the globular head and the 4HB stalks in the four-heads-
down arrangement (Fig. 6C). Interestingly, residues N121 and
N125 (PIV5 HN), in addition to their putative contacts in the
head–stalk interface of the ‘four-heads-down form (Fig. 6C), also
form contacts within the dimer-of-dimer interface of the PIV5
HN four-heads-up form (Fig. 6A). Unlike N121 and N125, P233
(PIV5 HN) is not involved in this dimer-of-dimer interface. To
test the role of these interfaces observed in the two different
structural arrangements obtained for PIV5 HN (16) and NDV
HN (15), mutations were made in the PIV5 HN residues N121A,
N125A, and P233L.
To analyze expression of the mutant HN proteins, transfected
cells were radiolabeled metabolically with [35S], and proteins
were immunoprecipitated. The expression levels of the mutants
were comparable to those of WT HN protein (Fig. 6 D and E).
The cell-surface expression levels of N121A, N125A, and P233L
mutants were comparable to WT HN, ranging between 70–110%
of WT PIV5 HN (Fig. 6F). The receptor-binding ability of these
mutants, determined by their ability to bind chicken RBCs
(hemadsorption), was variable. When normalized to surface ex-
pression, P233L has a normal receptor-binding capability, whereas
N121A and N125A have somewhat reduced receptor-binding
ability (Fig. 6G). However, N121A and N125A (part of both
head–stalk and dimer-of-dimer interfaces) were severely im-
paired for fusion activation, whereas P233L (part of the head–
stalk interface only) showed WT levels of fusion with PIV5 F
(Fig. 6H). There are two explanations for these data. One is that
altering the stalk–head interaction by N121A, N125A, or P233L
in the four-heads-down form does not affect fusion activation. In
this case, we assume that in the four-heads-up form, the N121A
and N125A mutations alter the dimer-of-dimer interface and
fusion activation. In contrast P233L does not disrupt an interface
in the four-heads up form, and fusion activation is observed. The
alternative explanation is that the mutations N121A and N125A
stabilize the head–stalk interaction and thus inhibit fusion.
The F protein of paramyxoviruses causes membrane fusion
through the concerted action of the F protein and the receptor-
binding protein HN, H, or G. The F protein folds initially to
form a trimeric metastable prefusion form that is triggered to
undergo large-scale irreversible conformational changes to form
the trimeric postfusion conformation. It is thought that F-protein
refolding couples the energy released with membrane fusion.
The irreversible nature of F-protein refolding requires a highly
specific mechanism so that triggering occurs only on receptor
binding by an attachment protein, i.e., HN, H, or G. The precise
mechanism by which HN, H, or G activates F is unclear.
It is well established for many paramyxoviruses that F and HN,
H, or G interact physically, as shown by coimmunoprecipitation
and cocapping studies (3, 25, 32, 41, 42). However, coimmuno-
precipitation of PIV5 F and HN has been difficult to demon-
strate; it may be a weak and/or transient interaction. The
available data suggest that the sites of F interaction reside within
the stalk domains of HN, H, or G (21–23, 25–34). The commonly
adopted hypothesis for F activation is that, on receptor binding,
the receptor-binding protein HN, H, or G undergoes structural
changes in the globular head that are transmitted through the
connecting loops to the stalks, where F interacts with the stalk
(20, 24, 35, 43). However, our finding of fusion promotion by
a headless PIV5 HN 1–117 stalk suggests that the F-activating
region residing within the HN 1–117 stalk is sufficient to activate
PIV5 fusion. Addition of carbohydrate chains to residues on the
outer surface of the PIV5 HN 1–117 stalk 4HB ablate fusion,
a finding indicative of an F protein–HN 1–117 stalk interaction.
of the chimeric protein PIV5-NDV-HN, consisting of the PIV5 HN stalk (amino
acids 1–117) followed by the NDV HN head (amino acids 124–571). (B) Sur-
face expression of PIV5-NDV-HN as measured by flow cytometry using
a mixture of NDV HN 4a and 2b mAbs and expressed as a percentage of WT
NDV HN expression. (C) Receptor-binding activity of PIV5-NDV-HN compared
with PIV5 HN and NDV HN and expressed as a percentage of WT NDV HN
bound to chicken RBCs at 4 °C. (D) Neuraminidase activity of the PIV5-NDV-
HN chimera compared with PIV5 HN and NDV HN proteins, as a measure of
fluorescent product formation, expressed as a percentage of WT NDV HN.
(E) Fusion promotion of the PIV5-NDV-HN chimera as measured by luciferase
reporter assays at 33 °C and 37 °C.
Chimeras and NDV HN stalk activation. (A) Schematic representation
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Furthermore, the PIV5 HN 1–117 stalk does not activate other,
noncognate F proteins.
Two lines of evidence indicate that the expression of PIV5 HN
1–117 stalk overcomes an energy barrier more readily than WT
HN. First, at 33 °C HN 1–117 stalk triggers fusion to a greater
extent than does WT HN. Second, HN 1–117 stalk triggers fu-
sion of the hypofusogenic mutant F-P22L orders of magnitude
more readily than WT HN. The explanation best supported by
the data suggests a distinct energy requirement by the globular
heads of PIV5 HN, possibly to undergo structural rearrange-
ments, whereas the HN 1–117 stalk may be able to activate F-
P22L because of an unlimited period of F–HN stalk interaction.
Similarly, in the hybrid PIV5-NDV HN construct, fusion was
greater at 37 °C than at 33 °C.
To expand the observation that PIV5 F can be activated by its
cognate HN stalk, we expressed various HN stalk constructs of
NDV and hPIV3 both with and without HA-epitope tags at their
N and C termini (Table S1). None of these HN stalk constructs
activated the cognate F protein for fusion. The addition of a HA-
tag to the C terminus of PIV5 HN 1–117 stalk reduced fusion
activity; expression of a PIV5 HN stalk that was shorter in length
(residues 1–110) and lacked the interchain disulfide bond also
reduced fusion. Because of the apparent loss of fusion activity
upon epitope-tagging the PIV5 HN 1–117 stalk and because of
the inability to detect hPIV3 and NDV HN protein stalks on the
cell surface using available antibodies, the F-triggering capa-
bilities of these proteins cannot be interpreted. As the mecha-
nism of F activation by the PIV5 HN 1–117 stalk, we propose
that the PIV5 HN 1–117 stalk in absence of the head domains
folds into a receptor-bound F-triggering 4HB conformation,
similar in concept to the structural rearrangements that occur on
the CDV H stalk with receptor binding (23). This F-triggering–
competent PIV5 HN stalk encounters F only on reaching the cell
surface (44) and triggers F in the absence of receptor binding.
Appending tags to or removing N-terminal residues from the
PIV5 HN 1–117 4HB may disrupt the F-triggering conformation,
causing the PIV5 HN 1–117 stalk to lose its F-triggering capa-
bility. For other paramyxoviruses the attachment protein heads
may be essential to stabilize the receptor-bound F-triggering
conformation, which does not occur with expression of the
The major question that arises is whether activation of fusion
by PIV5 HN 1–117 stalk is a mechanism that is unique to PIV5
or is related to processes occurring in other paramyxoviruses.
The atomic structure of NDV HN (15) indicates there is a flexi-
ble linker between the globular head domains and the 4HB of
the stalk, suggesting that the head domains can move. Different
positions of the PIV5 HN head domains are observed by electron
microscopy (Fig. 2E). The energy requirements for moving HN,
H, or G heads may vary among paramyxoviruses and may depend
on receptor engagement. Similarly, different paramyxoviruses
may differ in the flexibility and movement of the top of the stalk.
A recent study using engineered disulfide bonds between head
domains of the measles H dimer proposed possible conforma-
tional changes between monomers of this dimer that could in-
domains have important regulatory roles in fusion and receptor binding. (A)
Bottom view of the PIV5 HN four-heads-up form showing the positions of
N121 (green), N125 (green), and P233 (red) residues on all four monomers.
The dimer-of-dimer interface is indicated by a black box. (B) Side view of the
four-heads-down form as a structural alignment of PIV5 HN globular heads
(aquamarine) with the NDV HN structure (orange) showing the head/stalk
Structurally conserved loops in the paramyxovirus HN globular head
interface (black boxes). (C) Magnified top view of B. Corresponding PIV5 HN
residues (blue) and NDV HN residues (red) are highlighted in the head–stalk
interface. (D and E) Expression of PIV5 HN, N121A, and N125A (D) and P233L
mutant polypeptides (E) immunoprecipitated from radiolabeled cell lysates
with PIV5 HN pAb R471. (F) Surface expression of the PIV5 HN globular head
mutants expressed as a percentage of WT PIV5 HN surface expression using
PIV5 HN pAb R471 and flow cytometry. (G) Receptor-binding activity of
mutant proteins expressed as a percentage of WT PIV5 HN bound to chicken
RBCs at 4 °C. (H) Luciferase reporter assay for fusion showing fusion pro-
motion by the N121A, N125A, and P233L mutants cotransfected with PIV5-F,
expressed as a percentage of WT PIV5-F and WT PIV5 HN fusion.
Bose et al. PNAS Early Edition
| 7 of 10
fluence fusion promotion by measles virus H (19). However,
different results have been obtained in studies with NDV HN, in
which minor or no effects were observed on fusion using mutants
in which the globular head dimers were linked through disulfide
bridges (45). Additionally, the atomic structures of NDV HN
(15), PIV5 HN (16), and measles virus H bound to its cellular
receptor SLAM (20) support the idea that the dimer interfaces in
these attachment proteins remain intact; instead, the dimer-of-
dimer interfaces appear more prone to disruptions or changes.
Differences in fusion activation (Fig. 6H) and receptor-binding
abilities (Fig. 6G) in the dimer-of-dimer interface of PIV5 HN
N121A and N121A mutants suggest that maintaining a head
dimer in the upright position is essential for triggering fusion,
and the dimer-of-dimer interface in the PIV5 HN structure
creates an arrangement whereby both the dimers can attain a
heads-up conformation. On the other hand, P233L did not affect
fusion in the putative head–stalk interface of PIV5 HN, sug-
gesting that this contact may not be essential in the F-triggering
process. However, we cannot discount the possibility that N121A
and N125A mutations influence interactions in the head–
Syncytia formation by PIV5 F protein expressed alone has
been observed repeatedly at 37 °C, but it is greatly enhanced by
coexpression of PIV5 HN (Fig. 1C) (2, 46) or by incubation of
cells expressing F at 42 °C (Fig. 4 B and C). Hyperfusogenic
mutants of PIV5 such as F-S443P expressed alone can cause
syncytia formation at 25 °C (38). Taken together, these data
suggest that a major role of HN is to lower the energy barrier for
triggering metastable F. Syncytia formation without HN ex-
pression suggests either that another receptor molecule interacts
with F or that the close proximity of confluent tissue-culture cells
permits F to fuse without receptor engagement. An atomistic
model of pre-hairpin F predicts that the distance F can span is
210 Å (47). Syncytia formation mediated by respiratory syncytial
virus (RSV) and human metapneumovirus (hMPV) that lack G
has been well documented (48, 49), and both RSV and hMPV F
proteins have been shown to interact with glycosaminoglycans
and heparin sulfate, respectively (50, 51). Also, mutants of NDV
F and Sendai virus F have been made that are hyperfusogenic
and have a much lower need for HN (52, 53). Thus, it seems
possible that mutants of PIV5, NDV, and Sendai virus F that
lower the energy barrier do not require a receptor molecule for
syncytia formation in tissue-culture cells.
We propose a simple model for activation of fusion in para-
myxovirus (Fig. 7 A and B). The HN, H, or G attachment protein
stalk harbors the region for fusion activation, which remains
covered by the globular heads in the four-heads-down form (Fig.
7A). In this conformation, F is unable to interact with the F-ac-
tivating region of the attachment protein stalk. When receptor is
bound via the HN globular head regardless of whether there is
one sialic acid-binding site or two (NDV HN), energy is required
to move the heads to attain the four-heads-up form (Fig. 7B).
Because the heads can bind receptor at 4 °C, this energy probably
is required to expose the region of F activation on the stalk and to
allow F to interact with the stalk of the attachment protein to
The model we propose suggests a core mechanism of para-
myxovirus F activation by HN, H, or G proteins. However,
certain additional requirements and variations among paramyxo-
virus subfamilies exist (43). The atomic structures of measles virus
H and henipavirus G proteins (9, 10, 12, 14, 20) indicate that the
arrangement of the globular heads is somewhat different from
that of HN proteins, and the proteinaceous receptor is bound
by a lateral surface of the β-propeller, not at the top, where HN
binds sialic acid. Nonetheless, a degree of functional conserva-
tion in F activation among viruses binding different receptors is
apparent, especially in the stalk domain as found for the CDV H
protein (23). However, the morbillivirus H and henipavirus G
proteins require structural changes (20, 23) or a unique domain
(22) in their putative stalk 4HBs to activate their cognate F. No
effect on fusion was seen in experiments in which the length of
the measles H stalk was extended above the F-interacting region,
but insertions below this region abrogated fusion (25). These
data also argue against any F-triggering interaction with the HN,
H, or G head.
The site in the cell where F associates with the attachment
protein differs among paramyxoviruses. F–HN proteins do not
interact in the endoplasmic reticulum (ER) (44, 54), whereas F–H
proteins associate as complexes in the ER (55, 56). For this reason
the PIV5 HN 1–117 stalk, even though it lacks the regulatory head
domains, can traffic independently to the surface without trig-
gering F prematurely. Also measles virus H mutants have been
binding protein in the four-heads-down position. The formation of the contacts in the head–stalk interface prevents physical interaction of F with the stalk
region of the receptor-binding protein. (B) Creation of the contacts in the dimer-of-dimer interface of the receptor-binding protein in the four-heads-up
position moves the heads upwards, exposing the stalk. This exposure allows F to interact with the HN stalk, causing F triggering. Black balls indicate the sialic
acid binding site residues in the four neuraminidase head domains (colored red, green, blue, and yellow) of paramyxovirus HN. Prefusion F is represented by
the PIV5 prefusion F structure (purple) (6). Dotted lines represent regions of paramyxovirus F and HN proteins for which no structural data are available. CT,
Schematic model of paramyxovirus F activation based on putative structural rearrangements in the receptor-binding protein. (A) The receptor-
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| www.pnas.org/cgi/doi/10.1073/pnas.1213813109 Bose et al.
found to associate with F although unable to trigger fusion (28),
and a stronger F–G or F–H association leads to lower fusion ac-
tivity in henipaviruses and morbilliviruses (41, 58–59), suggesting
that F–G or F–H association, by itself, may not trigger F.
Given the positions of the heads in the measles tetramer, the
differences in stalk length between HN, H, or G proteins, the
different positions of receptor-binding sites on the globular heads
of H and G proteins, and conformational changes in the mor-
billivirus H stalk for F triggering, the prereceptor-bound forms of
morbillivirus or henipavirus stalks may differ significantly from
the four-heads-down form observed in NDV-HN. In addition, the
prereceptor-bound form of H or G may associate with F in
a complex before F triggering. However, we suggest that trig-
gering F upon receptor binding would require both structural
rearrangements between the HN/H/G globular head dimers to
expose the F-activating region and flexibility within the stalk 4HB
of the attachment protein to allow F interactions with the central
region of the HN, H, or G stalk domain, thus providing the core
trigger at the heart of the F-activation mechanism.
Cells and Antibodies. Vero, 293T, BHK-21F, and BSR-T7/5 (a BHK clone ex-
pressing T7 RNA polymerase) cells were grown as described (38). Antibodies
specific for PIV5 HN globular head included a mix of mAb HN-1b ascites fluid
and mAb HN-4b hybridoma supernatant (37). The PIV5 HN full-length and
stalk were detected using HN pAb R471, as described previously (21). PIV5 F
antibodies included mAb F1a ascites fluid (37), specific to prefusion cleaved
PIV5 F, and mAb 6-7, specific to the postfusion form of PIV5 F (60). NDV HN
mAbs 4a and 2b were cell-culture supernatants (40).
Cloning and Mutagenesis. pCAGGS-HN, pCAGGS-F, and pCAGGS-F P22L ex-
pression constructs harboring the PIV5 (W3A) HN, F, and F-P22L mutant genes,
respectively, were used as described previously (38). The PIV5 HN stalk con-
struct (1–117) was created by PCR amplification of the stalk region (residues
1–117) from the full-length PIV5 HN (residues 1–565). The PCR fragment then
was cloned into the pCAGGS vector. Mutagenesis of pCAGGS-HN or pCAGGS-
HN 1–117 stalk was done as described (21). The PIV5-NDV-HN chimeric protein
construct was amplified using four-primer PCR from pCAGGS-NDV-HN (Aus-
tralia-Victoria strain) and pCAGGS-PIV5 HN (strain W3A) and was cloned into
the pCAGGS vector. The nucleotide sequences were verified using an Applied
Biosystems 3100-Avant automated DNA sequencer (Life Technologies Corp.).
Expression of HN and F Glycoproteins in Mammalian Cells. PIV5 WT F and HN
(W3A strain) and their mutant proteins were expressed by transient trans-
fection using pCAGGS constructs in Vero, BHK-21F, and 293T cells.
293T cells, proteins were labeled metabolically with [35S] at 18 h posttransfec-
tion, and proteins were immunoprecipitated as described previously (21). Poly-
peptides were analyzed on 10% (wt/vol) or 15% (wt/vol) acrylamide gels.
Flow Cytometry. The cell-surface expression of PIV5 HN and its mutants was
quantified by flow cytometry using a FACSCaliber flow cytometer (Becton
Dickinson) as described previously (21). The PIV5 HN 1–117 stalk was detected
using the PIV5 HN pAb R471 at 1:100 dilution (21). To detect WT F or F-P22L
conversion to the postfusion form on the cell surface, 1:200 dilutions of
mAb F1a or mAb 6-7 were used. NDV HN monoclonal antibodies 2b and
4a were used at 1:10 dilution to detect chimeric proteins with NDV HN
Hemadsorption Assay and Neuraminidase Assay. Transfected 293T cell mon-
olayers were allowed to bind 1% chicken RBCs as described previously (38).
The neuraminidase activity of the PIV5 HN globular head mutants was de-
termined as described previously (21).
Syncytia Formation. Transfected BHK-21 cells were placed at 37 °C, 33 °C, or
42 °C to allow fusion. Eighteen hours posttransfection, the cells were
washed with PBS, fixed, and stained using a Hema3 staining protocol (Fisher
Scientific) according to the manufacturer’s instructions. For analyzing sialic
acid receptor engagement by WT HN and HN 1–117 during fusion, DMEM
containing C. perfringens neuraminidase (Sigma Scientific) or zanamivir was
added just after transfection, and cells were fixed and stained 18 h post-
transfection. A 50-mM stock solution of zanamivir was prepared from
Relenza Rotadisks (GlaxoSmithKline) (5 mg zanamivir with lactose) (61).
Luciferase Reporter Assay. To quantitate fusion observed in the syncytia assay,
Vero cell monolayers were transfected with 1 μg of pCAGGS-F, pCAGGS-HN,
or the HN mutant DNA and pT7-luciferase, a plasmid that expresses firefly
luciferase under T7 polymerase control. BSR-T7/5 cells were overlaid on the
Vero cell monolayer at 15 h posttransfection and were incubated further for
6–7 h at 37 °C or 33 °C. Cells (0.5 mL) were lysed with 2× Reporter Lysis Buffer
(Promega). Subsequently, the cell lysates were frozen overnight at −80 °C
to facilitate lysis and release of luciferase. Cell debris was pelleted from the
samples by centrifugation (2,000 × g for 10 min at room temperature), and
150 μL of the cleared lysates was added to a 96-well dish together with 150
μL of the luciferase assay substrate (Promega). The luciferase activity in rel-
ative light units then was determined using a SpectraMax M5 plate reader
Electron Microscopy. Solutions of PIV5 HN ectodomain (residues 56–565) (5 μg/
mL) were absorbed onto 300-mesh copper grids covered with a carbon film
that had been freshly glow discharged. Grids were stained with a 1%
aqueous solution of uranyl formate, and protein was observed in a JEOL
1230 electron microscope operated at 100 kV. Images were acquired with a
Gatan 831 CCD camera.
ACKNOWLEDGMENTS. The mAbs specific for Newcastle disease virus HN
protein were a generous gift from Ronald Iorio (University of Massachusetts
Medical School). This research was supported in part by National Institutes of
Health Research Grants AI-23173 (to R.A.L.) and GM-61050 (to T.S.J.). B.D.W.
was an Associate and R.A.L. is an Investigator of the Howard Hughes
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