JOURNAL OF VIROLOGY, Nov. 2006, p. 10931–10941
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 22
Characterization of Human Metapneumovirus F Protein-Promoted
Membrane Fusion: Critical Roles for Proteolytic
Processing and Low pH?
Rachel M. Schowalter, Stacy E. Smith, and Rebecca Ellis Dutch*
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0509
Received 19 June 2006/Accepted 29 August 2006
Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily
within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe
respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains
features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential
N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the
F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein
(G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be
analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined
the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate
that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein
does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when
transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may
function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and
three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate
endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are
utilized and that each affects cleavage and fusion to various degrees.
Human metapneumovirus (HMPV) was identified in 2001
by careful analysis of samples from children with respiratory
tract disease for which an etiological agent had not been iden-
tified (46). Since the discovery of HMPV, clinical studies have
demonstrated that it is a causative agent of respiratory tract
disease worldwide (17, 23, 27, 32, 38, 39), and examination of
serological samples indicates that HMPV has been circulating
in human populations since at least 1958 (46). Between 6 and
12% of children under age five with acute respiratory tract
infections are positive for HMPV (18, 47, 48), the clinical
symptoms of which closely resemble those seen with respira-
tory syncytial virus (RSV), ranging from coughing and wheez-
ing to bronchiolitis and pneumonia (9, 10, 24). The cytopathic
effect of HMPV has been reported to depend on the strain of
HMPV, with some strains inducing syncytium formation sim-
ilar to that of RSV, while other strains cause focal rounding
and cell destruction (20). Sequence analysis places HMPV in
the Pneumovirinae subfamily of the Paramyxoviridae family, as
it is closely related to avian pneumovirus, a cause of severe
respiratory infection in turkeys, as well as RSV, the most com-
mon cause of severe lower respiratory tract infection in infants
and the elderly (16, 45).
As it is an enveloped virus, entry of HMPV into host cells
requires the fusion of viral and cellular membranes. Paramyxo-
virus entry usually requires two viral glycoproteins, the fusion
(F) and attachment (G, H, or HN) proteins (2, 14), and mem-
brane fusion promoted by all paramyxovirus glycoproteins that
have been examined takes place at neutral pH, with one pos-
sible exception (15, 21, 25). The mechanism of SER virus
glycoprotein-induced fusion is controversial, as early studies
indicated a requirement for low pH (43), but recent work has
found efficient SER virus F protein-mediated membrane fu-
sion at neutral pH (8). In addition to virus-cell membrane
fusion, paramyxovirus glycoproteins also promote cell-cell fu-
sion. Multinucleated giant cells, termed syncytia, can be found
in tissues that have been infected by a variety of paramyxovi-
ruses (26, 34). Cultured cells infected with RSV form syncytia,
but examination of primary human airway epithelial cells in-
fected with RSV suggests that syncytium formation by this
virus may not be a common in vivo occurrence (50). Neverthe-
less, cell-to-cell fusion and the formation of syncytia are valu-
able tools for the study of fusion protein function. Expression
of viral glycoproteins alone in tissue culture cells is sufficient to
induce the formation of syncytia, thus permitting the careful
examination of glycoprotein function in the absence of any
other viral proteins.
The amino acid sequence of the HMPV F protein shares
33% sequence identity with the RSV F protein (45), and it
appears to contain each of the features characteristic of type I
viral fusion proteins (Fig. 1) (13). However, the HMPV F
protein is only 10 to 18% homologous with the F proteins of
paramyxoviruses outside the pneumovirus subfamily (45). The
HMPV G protein, the putative attachment protein, shares no
significant sequence similarity with other paramyxovirus at-
tachment proteins, although it contains a high content of
* Corresponding author. Mailing address: Department of Molecular
and Cellular Biochemistry, University of Kentucky College of Medi-
cine, Biomedical Biological Sciences Research Building, 741 South
Limestone, Lexington, KY 40536-0509. Phone: (859) 323-1795. Fax:
(859) 323-1037. E-mail: email@example.com.
?Published ahead of print on 13 September 2006.
serine, threonine, and proline residues, which is also a feature
of the RSV G protein (45). Whereas the attachment protein
plays a key role in fusion of the Paramyxovirinae subfamily of
the Paramyxoviridae family, its function in the Pneumovirinae
subfamily now appears to be different. Recombinant HMPV,
avian pneumovirus, and human and bovine RSV lacking the G
protein can enter cells and proliferate, suggesting that the
F protein alone can promote attachment and fusion (7, 31,
Despite this notable distinction between the two branches of
the Paramyxoviridae family, the F proteins of both appear to be
structurally similar. Paramyxovirus F proteins are trimeric type
I integral membrane proteins (13), and they are modified by
N-linked carbohydrates, which are often required for protein
folding or modulation of fusion (1, 11, 28, 29, 51). Further-
more, F proteins are synthesized as fusogenically inactive pre-
cursors (F0) that are subsequently cleaved to form a biologi-
cally active, disulfide-linked heterodimer, F1? F2(13, 22, 40).
The great majority of paramyxovirus F proteins are cleaved by
ubiquitous cellular proteases, such as furin or cathepsin L (35,
36). However, HMPV, like Sendai virus and human parainflu-
enza virus type 1, requires the addition of trypsin to efficiently
propagate in cell culture, presumably to cleave the F protein
(6, 46). Work performed by Schickli and coworkers suggests
that trypsin does indeed process the HMPV F protein into a
mature form (41). However, laboratory-expanded strains of
HMPV in which the F protein underwent trypsin-independent
cleavage were isolated, and the F protein residues that ap-
peared to contribute to trypsin-independent cleavage were
identified (41). Recently, the furin consensus sequence was
introduced into the HMPV F protein cleavage site of a recom-
binant virus in order to examine the effects of trypsin-indepen-
dent cleavage on tissue tropism and virulence (5). This F pro-
tein mutant was cleaved efficiently in the absence of trypsin,
but infection was still restricted to the respiratory tract.
We chose to examine cell-cell fusion promoted by HMPV
glycoproteins in transiently transfected cells in order to care-
fully dissect the basic requirements for membrane fusion pro-
moted by this virus. In addition, we made mutations proximal
to the putative F protein cleavage site and in potential N-
linked glycosylation sites in order to understand the posttrans-
lational modifications made to the HMPV F protein and the
effects of these mutations on fusion. Using syncytium and
luciferase reporter gene fusion assays, we show that the HMPV
F protein can promote fusion in the absence of the G protein
and that trypsin is indeed necessary to process the F protein
into a fusogenic form. Surprisingly, we also found that fusion
could be detected only when cells expressing the F protein
were exposed to low pH, suggesting that this viral fusion pro-
tein may function in a manner unique among the paramyxo-
MATERIALS AND METHODS
Cell lines. Vero cells, BHK cells, and BSR cells (provided by Karl-Klaus
Conzelmann, Max Pettenkofer Institut) were grown in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco Invitrogen, Carlsbad, Calif.) supplemented with
10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The media of
BSR cells was supplemented with 0.5 mg/ml G-418 sulfate (Gibco Invitrogen)
every third passage to select for the T7 polymerase-expressing cells.
Plasmids. HMPV F and G genes within the pGEM-3Zf(?) and pBluescript
SK(?) vectors, respectively, were kindly provided by Ursula J. Buchholz (NIAID,
Bethesda, Maryland). The HMPV F gene was released from pGEM-3Zf(?) and
ligated into the pCAGGS mammalian expression vector following digestion with
EcoR1 and Sph1. The HMPV G gene was released from pBluescript SK(?) and
ligated into the pCAGGS mammalian expression vector following digestion with
Sma1 and Xho1. HMPV G was moved from pBluescript SK(?) to the pGEM-4Z
vector, using Pst1 and Sal1 restriction digestion sites. All HMPV F protein mutants
were created while the gene was in pGEM-3Zf(?), using QuikChange site-directed
mutagenesis (Stratagene). Then the F genes with mutations were subcloned into
pCAGGS as described for the wild-type gene. All HMPV F, G, and mutant genes
used in this study were sequenced in their entirety. The plasmids pCAGGS-SV5 F
and pCAGGS-SV5 HN were provided by Robert Lamb (Howard Hughes Medical
Institute, Northwestern University).
Antibodies. Antipeptide antibodies (Genemed Synthesis, San Francisco,
Calif.) were generated using amino acids 12 to 26 of HMPV G and 524 to 538 of
Syncytium assay. Subconfluent monolayers of BHK or Vero cells in 6-well
plates were transiently transfected with a total of 2 ?g of DNA consisting of
pCAGGS-HMPV F (1 ?g) or an F protein mutant (1 ?g) and pCAGGS-HMPV
G (1 ?g) or the empty pCAGGS vector (1 ?g), using Lipofectamine Plus
reagents (Invitrogen) according to the manufacturer’s instructions. One excep-
tion is the “mock”-transfected BHK cells, which received 2 ?g of the empty
pCAGGS vector. The next morning, confluent cell monolayers were washed and
incubated at 37°C in Opti-MEM (Gibco) with 0.3 ?g/ml TPCK (L-1-tosylamide-
2-phenylethyl chloromethyl ketone)-trypsin for 1 to 2 h (0.2 ?g/ml TPCK-trypsin
for BHK cells). Then the cells were rinsed once with phosphate-buffered saline
(PBS; pH 7.2) before PBS of the indicated pH, buffered with 10 mM HEPES and
5 mM MES (2-(N-morpholino)ethanesulfonic acid hemisodium salt), was added.
Cells were incubated for 4 min at 37°C under the pH conditions indicated in the
figure, and then the media was replaced again with Opti-MEM with 0.3 ?g/ml
TPCK-trypsin. The pH pulse was repeated three more times (2 to 3 h apart)
throughout the day. Vero cells were incubated for either a few hours at 37°C or
overnight at 33°C in order to allow final cellular rearrangements to take place.
Digital photographs of syncytia were then taken with a Nikon Coolpix995 camera
mounted on a Nikon TS100 inverted phase-contrast microscope. Pictures of
BHK cells were taken the same day, and BHK cells were maintained in Opti-
MEM with TPCK-trypsin for only 1 h prior to each pH treatment. They were
maintained in DMEM plus FBS the rest of the time.
Reporter gene fusion assay. Vero cells in 6-cm dishes were transfected with
genes of viral glycoproteins or mutants, as indicated in the figure legends, and the
T7 control plasmid (Promega) containing luciferase cDNA under control of the
T7 promoter, using Lipofectamine Plus reagents (Invitrogen). The following day,
Vero cells in one 6-cm dish were lifted from the plate surface with trypsin,
resuspended in DMEM plus 10% FBS, and overlaid onto two 35-mm dishes of
BSR cells. For pH titration experiments (see Fig. 3C), three 6-cm dishes of Vero
cells were mixed and overlaid onto six 35-mm dishes, and each dish was then
treated with a different pH solution. BSR cells constitutively express the T7
polymerase; thus, upon membrane fusion and content mixing of effector and
target cells, luciferase is expressed. The combined cells were incubated at 37°C
for 30 min. The cells were then rinsed once with PBS (pH 7.2) before PBS of the
indicated pH buffered with 10 mM HEPES and 5 mM MES was added. The cells
were incubated for 4 min at 37°C under the indicated pH conditions, the media
was replaced again by Opti-MEM with 0.3 ?g/ml TPCK-trypsin, and the cells
FIG. 1. Schematic of the HMPV F protein. The F protein is pro-
teolytically processed into two fragments, F1and F2, which are disul-
fide linked. Arrows point to potential sites of N-linked glycosylation.
The amino acid sequence upstream of the cleavage site is indicated
along with the mutations made to this site. FP, fusion peptide; HRA
and HRB, heptad repeats A and B; TM, transmembrane domain; WT,
10932 SCHOWALTER ET AL. J. VIROL.
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VOL. 80, 2006 HMPV F PROTEIN-PROMOTED FUSION 10941