Bioscience Reports, Vol. 20, No. 6, 2000
MINI REVIEW
Virus Membrane Fusion Proteins: Biological Machines that
Undergo a Metamorphosis
Rebecca Ellis Dutch,1 Theodore S. Jardetzky,2 and Robert A. Lamb3,4
Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F
protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp,
and the influenza virus haemagglutinin, share a number of common features. All contain
multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be
fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion
peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion
proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the
N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat
forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structural elements in the processing and function of these viral
fusion proteins is discussed.
KEY WORDS: Membrane fusion; viral glycoprotein; heptad repeats; proteolytic
processing; glycosylation; oligomerization; fusion peptides.
INTRODUCTION
The fusion of viral envelopes with cellular membranes, an essential step mediating
entry of enveloped viruses into the host cell, is both a fundamental part of the viral
life cycle and a paradigm for other membrane fusion events. This important process
has been shown to be promoted by specific viral proteins such as the hemagglutinin
(HA) protein of influenza virus, the Env protein of HIV and the fusion (F) protein
of paramyxoviruses (reviewed in Hernandez et al., 1996). For many viruses, such as
influenza virus and Semliki forest virus, this fusion event occurs within the acidic
lumenal environment of the cellular endosome, where low pH triggers a conformational change in the fusion protein which eventually leads to membrane fusion.
However, for a large number of viruses, including the paramyxoviruses and retroviruses, fusion occurs at the surface of the cell and pH-induced conformational
changes do not appear to be involved. Later during the infectious cycle, a number
of these fusion proteins, including the F proteins from paramyxoviruses, are
1
Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky, 40536.
Department of Biochemistry, Molecular Biology and Cell Biology.
3
Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, 60208–3500.
4
To whom correspondence should be addressed; E-mail: [email protected]. Fax: (847) 491–2467.
2
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0144-8463兾00兾1200-0597$18.00兾0  2000 Plenum Publishing Corporation
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expressed at the plasma membrane of infected cells and can mediate fusion with
neighboring cells to form syncytia, or multinucleated giant cells. This cytopathic
effect can lead to tissue necrosis in ûiûo and may also be a mechanism of virus
spread.
Fusion proteins from a group of widely disparate viruses share several important common features that will be the focus of this review (Fig. 1). This group
includes the paramyxovirus F protein, the human and simian immunodeficiency
virus (HIV and SIV) gp160 protein, the Env protein of retroviruses, the Ebola virus
GP, and the influenza virus hemagglutinin (HA) protein. It should be noted that a
number of viral fusion proteins, including those from the togavirus, rhabdovirus,
and flavivirius families, do not fall within this class (for review see Hernandez et al.,
1996). Fusion proteins of the class which includes the influenza virus HA protein
and the paramyxovirus F protein must form homotrimers to be transported out of
the endoplasmic reticulum (reviewed in Doms et al., 1993) and in order to be biologically active (Wilson et al., 1981; Russell et al., 1994; Fass et al., 1996; Chan et al.,
1997). In addition, these fusion proteins contain multiple sites for addition of Nlinked carbohydrate chains. Addition of these chains is essential for proper folding
and function (Gallagher et al., 1992; Roberts et al., 1993; Bagai and Lamb, 1995).
The precursor form of these fusion proteins must then undergo proteolytic cleavage
to be biologically active (Homma and Ohuchi, 1973; Scheid and Choppin, 1974;
Lazarowitz and Choppin, 1975; reviewed in Hunter and Swanstrom, 1990; Klenk
and Garten, 1994). The resulting TM domain-containing subunits (the F1 subunit
of the paramyxovirus F; gp41 of HIV gp160; GP2 from the Ebola GP; the HA2
subunit of influenza virus HA) have at or near their new N-termini a hydrophobic
region, termed the fusion peptide, which has been shown to insert into the target
membrane during the fusion process (Asano and Asano, 1985; Hernandez et al.,
1997; Damico et al., 1998). Additionally, two 4–3 heptad repeat regions are present
in each of these fusion proteins, one near the fusion peptide and the other near the
TM domain (Chambers et al., 1990; Buckland et al., 1992). These heptad repeat
regions are believed to be important in promotion of membrane fusion, as mutations
within these domains frequently give rise to a fusion-deficient phenotype (Cao et al.,
1993; Chen et al., 1993; Sergel-German et al., 1994; Reitter et al., 1995) and peptides
corresponding to these regions have been demonstrated to block the fusion process
(Wild et al., 1992; Jiang et al., 1993a, b; Wild et al., 1994; Lu et al., 1995; Rapaport
et al., 1995; Lambert et al., 1996; Yao and Compans, 1996; Young et al., 1997;
Joshi et al., 1998; Munoz–Barroso et al., 1999). For this group of fusion proteins,
the atomic structure of the intact ectodomain region is only known for influenza
virus HA. Thus, for this review the elements of fusion proteins described above will
be the focus, with emphasis given to the well characterized influenza virus HA protein as well as the paramyxovirus fusion proteins.
REQUIREMENT FOR TRIMERIZATION AND GLYCOSYLATION
The trimeric form of the influenza virus HA protein was conclusively demonstrated by use of X-ray crystallography almost two decades ago (Wilson et al., 1981).
Subsequent analysis indicated that the HA protein assembles into oligomers with a
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Fig. 1. Comparison of the paramyxovirus F protein, influenza virus hemagglutinin, and the HIV-1 env
protein. FPGfusion peptide, HR1Gheptad repeat 1, HRSGheptad repeat 2, TMGtransmembrane
domain.
t1/2 of between 5 and 7 min (Copeland et al., 1986; Doms and Helenius, 1986;
Gething et al., 1986). For many other viral fusion proteins, initial experimental data
led to varying conclusions. The paramyxovirus fusion proteins were suggested to be
either tetrameric (Sechoy et al., 1987; Collins and Mottet, 1991) or trimeric (Russell
et al., 1994) based on biochemical evidence. The experimental evidence was also
unclear concerning the oligomeric state of the HIV env glycoprotein, with both
trimeric or tetramic states proposed (Earl et al., 1990; Weiss et al., 1990). Recent Xray crystal and NMR structure analysis of portions of the SV5 F protein (Baker et
al., 1999) and the HIV-env protein (Chan et al., 1997; Weissenhorn et al., 1997;
Caffrey et al., 1998) have shown conclusively that these proteins are trimeric. In
addition, numerous studies indicate that without proper folding into a trimeric state,
these proteins generally do not reach the cell surface, and thus cannot promote
membrane fusion (reviewed in Doms et al., 1993).
Glycosylation of the fusion proteins is extremely important for proper folding
and trimerization, but the requirement for the number of glycosylation sites varies
between proteins. For the influenza virus HA protein, subtypes H5 and H7, five or
more of the normal seven N-linked oligosaccharide chains are needed for intracellular transport (Gallagher et al., 1992; Roberts et al., 1993). The Env protein of HIV
is extensively glycosylated, and glycans make up approximately 50% of the mass of
the gp120 subunit (Geyer et al., 1988). The gp41 protein contains either four or five
potential glycosylation sites, at least two of which are required for fusion activity
(Fenouillet et al., 1993; Fenouillet and Jones, 1995; Perrin et al., 1998). For the F1
subunit of the SV5 F protein, removal of any one of the four glycosylation sites had
deleterious effects, ranging from partial delays in intracellular transport to severe
transport delays and acute instability of the F protein (Bagai and Lamb, 1995).
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Interestingly, the most deleterious effects were noted when the glycosylation site
within the TM-proximal heptad repeat was mutated.
PROTEOLYTIC PROCESSING
Proteolytic processing of the inactive precursor forms of the influenza virus HA
protein, HIV or SIV gp160, paramyxovirus F protein and the Ebola Gp protein is
required to form a biologically active molecule. In the case of the paramyxovirus F
protein, the precursor F0 molecule is cleaved to the disulfide linked subunits F1 and
F2, with proteolytic activation of F0 requiring the sequential action of two enzymes,
the host protease that cleaves at the carboxyl side of an arginine residue, and a host
carboxypeptidase that removes the basic residues. Some paramyxovirus F proteins,
including the SV5 F protein, the mumps virus F protein and the respiratory syncytial
virus (RSV) F protein, have multi-basic residues at the cleavage site. These proteins
undergo cleavage intracellularly during transport of the protein through the trans
Golgi network. Furin, a subtilisin-like cellular protease localized to the trans Golgi
network, has sequence specificity for cleavage of R–X–K兾R–R, and experimental
evidence indicates that it is capable of cleaving the F protein intracellularly (Ortmann et al., 1994). F proteins from paramyxoviruses such as Sendia virus have a
single basic residue at their cleavage site, and are generally not cleaved intracellularly. However, the F0 precursor that is expressed at the cell surface and incorporated
into released virions can be cleavage-activated by the addition of exogeneous protease (Homma, 1971; Scheid and Choppin, 1974). The importance of the cleavage
site for viral virulence and pathogenicity is readily seen in the case of the Newcastle
disease virus F protein. Strains with multi-basic residues in the cleavage site are
virulent strains which are readily disseminated through the host, as opposed to
strains with F0 molecules having single basic residues, which are avirulent and tend
to be restricted to the respiratory tracts where the necessary secreted protease can
be found (Nagai and Klenk, 1977).
Cleavage of the influenza virus HA protein is essential for the infectivity of the
influenza viruses (Klenk et al., 1975; Lazarowitz and Choppin, 1975). For the H5
and H7 avian strains, cleavage is thought to occur by the action of the furin or PC6
proteases (Klenk and Garten, 1994). Other avian, and all equine, porcine and human
influenza virus strains, in contrast, have a more restricted host range, requiring cells
that secrete a protease which can cleave the HA0 precursor extracellularly. In the
case of the H1, H2 and H3 subtypes, this cleavage may be mediated by the tryptase
Clara, first isolated from rat bronchiolar epithelia cells (Kido et al., 1992). In tissue
culture systems, all of these strains require addition of exogenous trypsin to allow
growth. The notable exception to this rule is the influenza virus strain A兾WSN兾33
(Choppin, 1969), where serum plasminogen had been suggested to play a role in
cleavage of the WSN HA protein in tissue culture (Lazarowitz et al., 1973). Recently,
a novel mechanism for cleavage was proposed for the WSN strain. Plasminogenmediated HA cleavage was shown to occur only in the presence of the WSN neuraminidase (NA) protein (Goto and Kawaoka, 1998). The authors suggested that loss
of a carbohydrate chain at residue 146 of the WSN NA allows the WSN NA protein
to bind plasminogen, through an interaction with the NC C-terminal lysine residue,
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leading to cleavage of the plasminogen precursor to the active form plasmin. Plasmin
is then present in the proper place and in high relative concentration, and cleaves
the WSN HA protein. This unique cleavage mechanism could play a role in disease
processes.
The atomic structures of both the uncleaved and cleaved forms of the influenza
virus HA protein have been determined (Wilson et al., 1981; Chen et al., 1998). In
the uncleaved precursor form, the cleavage site is a prominent loop on the surface
adjacent to a cavity. Upon proteolytic cleavage, structural rearrangements occur
such that the newly created hydrophobic N-terminus is buried within the cavity,
while the vast majority of the HA protein remains unchanged. Structural information on the effects of cleavage on the HIV-1兾SIV gp160, Ebola virus GP or the
paramyxovirus F protein is not presently available. However, several lines of evidence suggest that the paramyxovirus F protein undergoes more dramatic conformational changes subsequent to proteolytic activation. For the Sendai virus F
protein, the α -helicity, as determined by circular dichroism, and the hydrophobicity,
as judged by Triton X-100 binding, were both found to increase after proteolytic
cleavage (Hsu et al., 1981). The NDV–F protein was found to undergo changes
in isoelectric point subsequent to cleavage (Kohama et al., 1981) and monoclonal
antibodies were shown to preferentially immunoprecipate the cleaved form of the
protein (Umino et al., 1990). It has also been shown that only the cleaved form of
the measles virus F protein gives rise to immunosuppression, leading to the suggestion that cleavage results in exposure of immunosuppressive domains (Weidmann et
al., 2000). Finally, major changes in recognition by a panel of anti-peptide antibodies
were found after cleavage of the SV5 F0 protein to the fusogenically active disulfide
linked heterodimer F1 and F2, with antibodies directed against the heptad repeat
regions recognizing only the uncleaved form (Dutch et al., 2001).
FUSION PEPTIDES
After precursor cleavage, the resulting TM domain-containing subunits have at
or near their new N-termini a hydrophobic region that is termed the fusion peptide.
These regions have been extensively studied, and more comprehensive reviews are
published within this volume. Briefly, for the paramxyoviruses, protein sequencing
studies of the F protein and nucleotide sequencing studies of the F genes showed
that the fusion peptide, which is thought to comprise the N-terminal 25 residues of
F1, is both extremely hydrophobic and highly conserved within the paramyxovirus
family, with up to 90% identity (reviewed in Lamb and Kolakofsky, 1996). The
fusion peptide regions are thought to intercalate into target membranes, thus initiating the fusion process, and evidence for this direct insertion into target bilayers has
been obtained using hydrophobic photoaffinity labeling probes (Novick and
Hoekstra, 1988). In addition, the fusion peptide from the paramyxovirus SV5 F
protein has been shown to be sufficiently hydrophobic that it can act as a TM anchor
domain to convert a formerly soluble protein to a membrane-bound form (Paterson
and Lamb, 1987). However, it seems likely that the fusion peptides play a more
complex role than simple insertion into the target membrane, as the fusion peptides
of the paramyxovirus F proteins contain many invariant residues, but neither signal
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sequences nor membrane anchorage domains show sequence conservation beyond
their hydrophobic nature. Mutations within the fusion peptide also suggest a more
complex role: when conserved glycine residues at positions 3, 7 or 12 were changed
to alanine, expression of the altered F proteins led to a dramatic increase in syncytium formation over that seen with the wild type F protein (Horvath and Lamb,
1992). If the fusion peptide is assumed to be an α -helix, then the invariant residues
are located on one face of the helix (Baker et al., 1999) again suggesting a specific
role of the fusion peptide residues in mediating the fusion reaction.
HEPTAD REPEAT REGIONS
Heptad repeat regions are present in the paramyxovirus F1 subunit, the HIV
and SIV gp41 proteins, the GP2 from the Ebola virus and the HA2 subunit from
influenza virus near both the fusion peptide and the TM domain (Chambers et al.,
1990; Buckland et al., 1992), and mutations within these domains often lead to a
fusion-deficient phenotype (Cao et al., 1993; Chen et al., 1993; Sergel-German et al.,
1994; Reitter et al., 1995). These two 4–3 heptad repeat regions, which in general
are predicted to promote coiled-coil interactions, were first shown to interact in a
fusion protein from the crystallographic analysis of the low pH form of the influenza
virus HA protein. The N-terminal heptad repeat was found to form a trimeric coiledcoil, with the C-terminal repeat regions forming helical regions that buttressed the
coiled-coil in an anti-parallel orientation (Bullough et al., 1994; Chen et al., 1999).
Biochemical data from the corresponding heptad repeat regions of the Moloney
murine leukemia virus (MoMLV) TM protein (Fass and Kim, 1995), the HIV and
SIV gp41 proteins (Blacklow et al., 1995; Lu et al., 1995; Weissenhorn et al., 1996),
the Ebola virus GP (Weissenhorn et al., 1998a), and the paramyxovirus fusion proteins SV5 F (Joshi et al., 1998; Dutch et al., 1999) and respiratory syncytial virus
(RSV) F (Matthews et al., 2000) all pointed to formation of a similar complex
between these heptad repeat regions. NMR and X-ray crystallographic analyses of
portions of HIV-1兾SIV gp41 (Chan et al., 1997; Tan et al., 1997; Weissenhorn et
al., 1997; Caffrey et al., 1998), a portion of a retrovirus Env-TM domain (Fass et
al., 1996), the majority of the Ebola virus GP2 (Weissenhorn et al., 1998b; Malashkevich et al., 1999) and peptides derived from the heptad repeats of the SV5 F
protein (Baker et al., 1999) confirm the biochemical data. In all cases, the N-terminal
heptad repeat regions form an interior, trimeric coiled-coil surrounded by three antiparallel helices from the C-terminal heptad repeat region (Fig. 2). In addition, the
N-terminal heptad repeat regions from the gp21 protein of human T cell luekemia
virus have been shown crystallographically to form an interior trimeric coiled-coil,
though in this case the C-terminal region packs in an extended anti-parallel conformation (Kobe et al., 1999). The similarity of these structures strongly suggests a
conservation of fusion mechanism, with an ancestral fusion protein related to each
(Baker et al., 1999).
Though many elements were similar among the atomic structures, several key
differences exist between the structure seen for the SV5 F protein and those seen for
the other above described fusion proteins. First, the structure of the F protein core
trimer extends to within 20 residues of the N-terminus of F1, and therefore includes
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Fig. 2. Comparison of the core trimer structures from the SV5 F protein (Baker et al.,
1999), the low-pH induced influenza virus HA, tBHA2 (Chen et al., 1999), HIV gp41
(Chan et al., 1997), MMLV Env–TM protein (Fass et al., 1996), and the Ebola virus
Gp (Weissenhorn et al., 1998a).
portions of what is generally considered the fusion peptide. Conserved residues
within the fusion peptide align with the heptad repeat of the interior coiled coil.
Thus, it has been hypothesized that the interior coiled coil of the SV5 F protein
extends up through the fusion peptide (Baker et al., 1999). This does not appear to
be the case for the other fusion proteins, as there are between 27 and 40 residues
between the first observed amino acid in the structure and the N-terminus of the
fusion peptide (Bullough et al., 1994; Fass et al., 1996; Weissenhorn et al., 1997;
Caffrey et al., 1998; Weissenhorn et al., 1998b; Chen et al., 1999). In addition, for
influenza virus HA the core trimer N-terminal heptad region is terminated by a cap
structure making it unlikely that the α -helix extends continuously to the fusion peptide (Chen et al., 1999).The second key difference between the structure seen for
SV5 F protein and other fusion proteins is that only seven amino acids are present
between the end of the C-terminal heptad repeat and the putative start of the transmembrane domain in the SV5 F protein. Mutational analysis has demonstrated that
these residues are dispensible for promotion of membrane fusion (Zhou et al., 1997;
Baker et al., 1999). In contrast, the structures of the HIV兾SIV gp41 and MMLVEnv-Tm proteins terminate 18 and 39 residues, respectively, from the putative TM
domain (Fass et al., 1996; Chan et al., 1997; Tan et al., 1997; Weissenhorn et al.,
1997; Caffrey et al., 1998) and in the structures of tBHA2 and the Ebola virus GP2,
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14 to 23 residues are disordered at the C-terminus (Bullough et al., 1994; Weissenhorn et al., 1998b). Finally, the paramyxovirus F protein membrane proximal subunit (F1) differs from that of HA2, gp41, Env-TM and GP2 because its ectodomain
is much larger in size (383 residues vs 120–185 residues), with ∼250 residues separating the heptad repeat regions.
CONFORMATIONAL CHANGES: PROMOTION OF MEMBRANE FUSION
What role do these structural elements play in promotion of membrane fusion? A
large body of evidence suggests that viral fusion proteins undergo a conformational
change to become fusion active (reviewed in Hernandez et al., 1996). For influenza
virus HA, proteolytic cleavage leads to a metastable conformation that is trapped
from achieving its lower-energy fusogenic confirmation by a kinetic barrier. During
virus uncoating in endosomes, the low pH within the endosomal lumen triggers a
conformational change in the HA protein, in which two separate α -helical regions
in the HA2 protein (Wilson et al., 1981) refold to form a single triple-stranded coiled
coil (Bullough et al., 1994; Chen et al., 1999). This results in the relocation of the
fusion peptide by ∼100 Å to one end of the rod-shaped coiled coil (Bullough et al.,
1994), as predicted by the hypothesis of thě spring loaded mechanism of fusion (Carr
and Kim, 1993). This conformation would allow insertion of the fusion peptide into
the target membrane (Fig. 3(A). In addition, HA residues 106–112, which are helical
in the neutral pH form of the HA protein, convert to a loop in the low pH-induced
form, leading to a bending of the protein, and the subsequent antiparallel orientation
of the heptad repeat regions (Bullough et al., 1994). This orientation places the
fusion peptide and TM domains on the same end of the molecule, and therefore
would facilitate the bringing together of the two lipid bilayers. As flexibility appears
to exist between the heptad repeat complex and the fusion peptide and TM domains
in the influenza virus HA protein, it is unclear whether membrane fusion occurs
during or after these conformational changes (reviewed in Skehel and Wiley, 1998;
Skehel and Wiley, 2000).
Though direct evidence for a triggering event or conformational changes under
fusogenic conditions is lacking for the paramyxovirus F proteins, the similarity of
the SV5 F1 heptad repeat core complex to the low-pH triggered confirmation of
the influenza virus HA protein strongly indicates some conservation of mechanism.
However, initiation of membrane fusion is clearly quite different: not only does the
paramyxovirus F protein function at neutral pH, but also, unlike influenza virus
HA protein and HIV gp160, the paramyxovirus F protein does not provide the
primary binding role for the virus. Instead, paramyxovirus primary binding to a
target cell is mediated by the hemaglutinnin-neurominidase (HN) glycoprotein.
While the SV5, measles virus, and RSV F proteins are capable of promoting membrane fusion in the absence of their homotypic HN protein (Paterson et al., 1985;
Olmsted et al., 1986; Alkhatib et al., 1990; Horvarth et al., 1992; Alkhatib et al.,
1994; Kahn et al., 1999), F proteins from many other paramyxoviruses require their
homotypic HN protein for promotion of membrane fusion (Sakai and Shibuta,
1989; Ebata et al., 1991; Morrison et al., 1991; Taylor et al., 1991; Wild et al., 1991;
Horvath et al., 1992; Hu et al., 1992; Cattaneo and Rose, 1993; Yao et al., 1997).
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Fig. 3. Two models of virus-mediated membrane fusion (A). A model based on
studies of the influenza virus HA and HIV gp41 crystal structures (Bullough et al.,
1994; Weissenhorn et al., 1997, 1998b; Chan and Kim, 1998). This model includes
two conformational transitions of the fusion protein, the insertion of the fusion
peptide into the target membrane, followed by refolding and juxtaposition of the
target and viral bilayers. Flexible linkers between the protein and the two membranes allow the coiled coil to reorient with its long axis parallel to the membrane
surface. Membrane fusion may occur during or after the conformational rearrangements by an unknown mechanism. (B) A model for paramyxovirus-mediated membrane fusion. Conformational events potentially analogous to those observed for
influenza virus HA are thought to lead to the insertion of the fusion peptide into
the target membrane. In contrast to the fusion model shown in the top row, the
structural and biochemical evidence suggest that flexible tethers to the two lipid
bilayers are not required for membrane fusion. Flexibility and the close approach
of the two membranes could instead be promoted by the intervening 250 amino
acids between the N- and C-terminal heptad repeats. This model would predict that
the free energy associated with conformational rearrangements of the fusion protein could be directly coupled to the fusion of the two membranes.
It has been proposed that the HN and F proteins physically interact and biochemical
evidence for such an HN兾F interaction has been obtained (reviewed in Lamb, 1993).
A model that would rationlize the involvement of the HN protein in promotion of
membrane fusion is that the hypothesized initial conformational change in the F
protein, leading to release of the fusion peptide, is highly regulated. For those F
proteins that require HN and F co-expression to give observable fusion, the first
step would be the binding of the HN protein to its receptor, sialic acid. This binding
event would cause the HN protein to undergo its own conformational change, which
in turn could trigger are conformational change in the F protein, leading to release
of the fusion peptide and initiation of membrane fusion. Examination of mutants of
the SV5 F protein that require coexpression of the HN protein to promote membrane fusion demonstrated that the HN function for F protein triggering can be
replaced by providing energy by raising the temperature (Paterson et al., 2000).
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Furthermore, raising the temperature also triggers the Sendai virus F protein to
initiate fusion (Wharton et al., 2000). This suggests that the F兾HN interaction provides energy to convert cleaved F from a metastable form to a fusogenic form (Paterson et al., 2000). Presumably, paramyxovirus F proteins that do not require their
homotypic HN protein for initiation of membrane fusion have a decreased energy
requirement for transition from their metastable state. It should be noted that Calder
and co-workers have observed two forms of the RSV F protein in the absence of the
RSV G protein by use of electron microscopy. They hypothesize that the coneshaped rods present in freshly prepared samples may represent a pre-activated state
of the F protein, while the lollipop-shaped rods that form upon storage may correspond to the fusion active state (Calder et al., 2000). As no G protein is present, the
observed structural changes must be able to occur independent of the G protein.
There are compelling reasons to believe that the paramyxovirus F proteins reach
a state that contains the heptad repeat cone complex only after one or more conformational changes. Major changes in recognition by a panel of anti-peptide antibodies were found after cleavage of the SV5 F0 protein to the fusogenically active
disulfide linked heterodimer F1 and F2, with antibodies directed against the heptad
repeat regions recognizing only the uncleaved form (Dutch et al., submitted). These
data strongly indicates that the heptad repeat complex is not present in the uncleaved
precursor form of the F protein, and that conformational rearrangements occur
subsequent to cleavage. This cleavage event likely places the F protein in a metastable form analogous to the influenza virus HA protein, with the fusion peptide
buried in this conformation of the cleavage-activated form to prevent aggregation
of the hydrophobic fusion peptides in an aqueous environment. Subsequent promotion of membrane fusion is initiated either by interaction with the paramyxovirus
HN proteins, or by other still to be determined triggering factors, resulting in release
of the fusion peptide and insertion of the fusion peptide into the target membrane
(Novick and Hoekstra, 1988; Fig. 3(B). Several lines of evidence indicate that the
conformation of the F protein which includes the heptad repeat core complex forms
only during the membrane fusion event. Membrane fusion can be blocked by the
addition of peptides corresponding to the heptad repeat regions (Rapaport et al.,
1995; Lambert et al., 1996; Yao and Compans, 1996; Young et al., 1997; Joshi et
al., 1998). This has been suggested to be due at least partially to their ability to
prevent formation of the heptad repeat core complex, indicating that this complex
does not form prior to the membrane fusion event. Perhaps the most compelling
data for a conformational change leading to the formation of the heptad repeat core
complex only during the membrane fusion event comes from the structure of the
paramyxovirus heptad repeat core complex and the data obtained with deletion
mutants in the F1 protein (Baker et al., 1999). No requirement for extended, flexible
linker regions between the F protein heptad repeat core complex and the fusion
peptide and TM domains is apparent. Indeed, increasing the spacing between the
heptad repeat complex and the TM domain by insertion of two, four, or six residues
inactivates the F protein for fusion (Zhou et al., 1997). This absence of flexibility
indicates that the fusion peptide and TM domains must be in extremely close proximity once the core complex has formed, a finding that is physically inconsistent
with a perfusion state in which the target and viral membranes are separate. Thus,
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for the paramyxovirus F proteins, it has been proposed that the free energy associated with conformational rearrangements of the fusion protein and formation of the
heptad repeat core complex could be directly coupled to fusion of the two membranes (Baker et al., 1999; Fig. 3(B).
For HIV-1 gp120/gp41 and retrovirus Env-Su+Env-TM, binding to the viral receptor
(and co-receptors, in the case of HIV-1) appears to induce the conformational change to
release the fusion peptide (Hernandez et al., 1997; Damico et al., 1998; Furuta et al., 1998).
The presence of the fusion peptide, the heptad repeat core structure, and the flexibility
between this core complex and the membrane bound regions are all similar to those seen in
the influenza virus HA protein, and suggest that these fusion proteins share a similar mechanism. However, it should be noted that peptides corresponding to the heptad repeat regions
of gp41 are potent inhibitors of membrane fusion (Wild et al., 1992; Jiang et al., 1993a;
Wild et al., 1994; Munoz–Barroso et al., 1998), as is seen for the paramyxovirus F proteins,
presumably by blocking formation of the heptad repeat complex. Recent studies on the
timing of inhibition by both these peptides and the positive spontaneous curvature lipid
lysophosphatidylcholine have led to the conclusion that the heptad repeat core complex of
the HIV-1 gp41 protein does not form prior to membrane merger (Melikyan et al., 2000).
Thus, it appears likely that the proposed model for paramyxovirus fusion (Fig. 3(B)) as
promoted by the formation of the heptad repeat core complex applies also to fusion by the
gp41 protein.
PERSPECTIVES
The past five years have yielded exciting results in the field of viral membrane
fusion proteins, including the elucidation of atomic structures of portions of fusion
proteins from several divergent viral families, and the subsequent identification of a
common core structure. The use of this knowledge to explore inhibitors of viral
fusion proteins (Eckert et al., 1999; Ferrer et al., 1999) represents a promising new
means of targeting a wide group of viruses. Future work should more clearly delineate the role of this core structure in promotion of membrane fusion, and also identify any other conformations of these viral fusion proteins. For the paramyxovirus F
proteins, the function of the large intervening region between the heptad repeat
regions remains a mystery which will likely be at least partially clarified in the coming years. Each finding increases our understanding of these biological machines,
leading us closer to a detailed understanding of their mechanisms.
ACKNOWLEDGMENTS
This work was supported by Research Grant AI-23173 from the National Institute of Allergy and Infectious Disease. R.E.D. was supported by Public Health Service NRSA F32 AI-09607. T.S.J. is a Pew Scholar and R.A.L. is an Investigator of
the Howard Hughes Medical Institute.
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