Thermostability of the human respiratory syncytial
virus fusion protein before and after activation:
implications for the membrane-fusion mechanism
M. Begon ˜a Ruiz-Argu ¨ello,13 Diana Martı ´n,13 Steve A. Wharton,2
Lesley J. Calder,2Steve R. Martı ´n,2Olga Cano,1Miguel Calero,1
Blanca Garcı ´a-Barreno,1John J. Skehel2and Jose ´ A. Melero1
Jose ´ A. Melero
1Centro Nacional de Microbiologı ´a, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid,
2National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
Received 26 May 2004
Accepted 3 September 2004
Anchorless fusion (F) proteins (FTM”) of human respiratory syncytial virus (RSV) are seen by
electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites
required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped
spikes following cleavage. To show that this aggregation occurred by interactions of the fusion
peptide, a deletion mutant of FTM”lacking the first half of the fusion peptide was generated.
This mutant remained unaggregated even after completion of cleavage, supporting the notion
that aggregation of FTM”involved the fusion peptide. As exposure of the fusion peptide is a key
event that occurs after activation of F proteins, the uncleaved and cleaved forms of FTM”may
represent the pre- and post-active forms of RSV F protein. In an analysis of the structural
differences between the two forms, their thermostability before and after proteolytic cleavage
was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza
haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of FTM”were equally
resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody
binding. These results are interpreted in terms of the proposed structural changes associated
with the process of membrane fusion mediated by RSV F protein.
Human respiratory syncytial virus (HRSV) is an enveloped,
non-segmented negative-stranded RNA virus, classified
within the genus Pneumovirus of the family Paramyxo-
viridae (Collins et al., 2001). It is the main cause of severe
lower respiratory tract infections in very young children
(Glezen et al., 1986) and is a pathogen of considerable
et al., 1995). The virion has two main surface glycoproteins:
the attachment (G) protein (Levine et al., 1987), which
mediates virus binding to cells, and the fusion (F) protein
(Walsh & Hruska, 1983), which is responsible for fusion
of the viral and cellular membranes. The F protein also
promotes fusion of the membrane of infected cells with
that of adjacent cells to form characteristic syncytia. A
third, small hydrophobic (SH) glycoprotein of unknown
function is expressed abundantly in infected cells, but is
incorporated only in small amounts into the virus particle
(Collins & Mottet, 1993). The G protein of HRSV shares
neither sequence nor predicted structural features with the
attachment protein of related viruses (Wertz et al., 1985). In
contrast, the F protein shares structural elements with its
counterparts in other paramyxoviruses and all F proteins
have a low, but significant level of sequence relatedness
(Collins et al., 1984).
The HRSV F protein is a type I glycoprotein that is
synthesized as an inactive precursor (F0) of 574 aa. This
precursor is cleaved by furin-like proteases during matura-
tion to yield two disulfide-linked polypeptides, F2 from
the N terminus and F1 from the C terminus. In contrast to
other paramyxovirus F proteins that are cleaved only once,
the F0 precursor of HRSV and the related bovine RSV are
cleaved twice, after residues 109 (site I) and 136 (site II),
which are preceded by furin-recognition motifs (Gonza ´lez-
Reyes et al., 2001; Zimmer et al., 2001) (see Fig. 1a for a
diagram of the primary structure). The F proteins of all
paramyxoviruses have three main hydrophobic regions:
one at the N terminus, which acts as the signal peptide for
translocation into the ER; another region, the membrane
anchor or transmembrane domain, near the C terminus;
and a third region at the N terminus of the F1 chain, called
the fusion peptide because it is thought, by analogy with
3These authors contributed equally to this work.
0008-0318 G 2004 SGM Printed in Great Britain3677
Journal of General Virology (2004), 85, 3677–3687
other fusion peptides (Durrer et al., 1996), to be inserted
into the target membrane during the process of membrane
fusion. The mature F protein is a homotrimer in which
two heptad-repeat sequences, HRA and HRB, adjacent to
the fusion peptide and to the transmembrane region of
each monomer, respectively, are important motifs for the
formation and stability of the trimers. HRA and HRB
peptides form trimeric complexes in solution (Lawless-
Delmedico et al., 2000; Matthews et al., 2000) and X-ray
crystallography of these complexes reveals an internal core
of three HRA a-helices bounded by three antiparallel HRB
a-helices, packed into the grooves of the HRA coiled-coil
trimer (Zhao et al., 2000).
HRSV enters the cell by fusion at the plasma membrane
rather than by endocytosis (Srinivasakumar et al., 1991).
There have been reports that the G and SH proteins enhance
the formation of syncytia mediated by the F protein when
the three proteins are expressed in the same cell (Heminway
et al., 1994; Pastey & Samal, 1997). It was thus assumed that
the G and SH proteins could also enhance fusion of the
viral and cellular membranes mediated by the F protein.
However, spontaneous mutants (Karron et al., 1997) or
genetically engineered recombinants of HRSV that lack G
and/or SH (Bukreyev et al., 1997; Techaarpornkul et al.,
2001) can still infect certain cell types and induce the
Fig. 1. Trypsin digestion of FTM”. (a) Diagram of the F protein primary structure, denoting hydrophobic regions (filled boxes),
heptad repeats (hatched boxes), cysteine residues ($), glycosylation sites (m) and sites of proteolytic processing (Q). A
diagram of the anchorless form of F (FTM”), which lacks the C-terminal 50 aa, is shown below. (b) Partial sequences, including
furin cleavage sites I and II (in bold) and the fusion peptides (italics), of wild-type FTM”and the D137–146 mutant. These two
proteins were purified by immunoaffinity chromatography as indicated in Methods. Aliquots (~0?2 mg) of each protein were
either digested with 0?2 mg trypsin/TPCK at 46C for 1 h (+) or mock-digested (”). The reaction was stopped by the addition
of sample buffer and boiling for 5 min. The proteins were resolved by SDS-PAGE and immunoblotted with antisera raised
against synthetic peptides aa 255–275 or 104–117, as indicated. Diagrams corresponding to the bands detected in the
immunoblot are shown on the right.
3678Journal of General Virology 85
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RSV F protein thermostability and membrane fusion