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Bioscience Reports, Vol. 20, No. 6, 2000
MINI REVIEW
Functional Domains within Fusion Proteins: Prospectives for
Development of Peptide Inhibitors of Viral Cell Fusion
Yechiel Shai1
Receiûed August 1, 2000
The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope
and target plasma membrane and is mediated by fusion proteins. Recently, several functional domains within fusion proteins from different viral families were identified. Some
are directly involved in conformational changes after receptor binding, as suggested by the
recent release of crystallographically determined structures of a highly stable core structure
of the fusion proteins in the absence of membranes. However, in the presence of membranes, this core binds strongly to the membrane’s surface and dissociates therein. Other
regions, besides the N-terminal fusion peptide, which include the core region and an internal
fusion peptide in paramyxoviruses, are directly involved in the actual membrane fusion
event, suggesting an ‘‘umbrella’’ like model for the membrane induced conformational
change of fusion proteins. Peptides resembling these regions have been shown to have
specific antiviral activity, presumably because they interfere with the corresponding
domains within the viruses. Overall, these studies shed light into the molecular mechanism
of membrane fusion induced by envelope glycoproteins and suggest that fusion proteins
from different viral families share common structural and functional motifs.
KEY WORDS: Membrane fusion, peptide–lipid interaction, fluorescence, viral fusion
inhibitors, antiviral peptides.
INTRODUCTION
The entry of enveloped viruses into host cells is accomplished by fusion of the viral
envelope and target plasma membrane (Hoekstra and Kok, 1989; Stegmann et al.,
1989; White, 1990). Membrane fusion is mediated by several glycoproteins present
in the viral envelope which help to overcome strong repulsive hydration, steric, and
electrostatic barriers. One of them, the fusion protein, is directly involved in the
membrane fusion event, i.e., the actual mixing of the viral and cell membranes.
Fusion proteins from different viral families (e.g. Paramyxo-, Orthomyxo-, and
Retroviridae) share conserved features (White, 1990). Specifically, (i) they are type
I integral membrane proteins synthesized as inactive precursors that are cleaved by
host-cell proteases to become active; (ii) the newly generated N-terminus contains
the fusion peptide, an hydrophobic stretch of amino acids believed to insert and
destabilize the membrane, leading to fusion (Durell et al., 1997; Epand, 1998); and
1
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail:
[email protected]
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0144-8463兾00兾1200-0535$18.00兾0  2000 Plenum Publishing Corporation
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Shai
(iii) either an heptad repeat adjacent to the N-terminal fusion peptide folds into
a protease-resistant trimeric coiled-coil at a certain step during the fusion process
(Stegmann et al., 1989; White, 1990; Skehel and Wiley, 1998), or two heptad repeats,
one adjacent to the N-terminal fusion peptide and the other to the C-terminal transmembrane domain fold into a trimer of heterodimers such that the interior contains
the N-terminal heptad repeat trimer and the outer ring the C-terminal heptad repeats
(Fass et al., 1996, Skehel and Wiley, 1998; Baker et al., 1999). However, there are
also exceptions to these common features among viral fusion proteins, since no
coiled-coils regions have been detected in the E envelope glycoprotein from flavivirus
(Rey et al., 1995).
ARE THE KNOWN 3D STRUCTURES THE ‘‘FUSION ACTIVE’’
CONFORMATIONS?
The recent release of crystallographically determined structures of fragments of
the fusion proteins of several viruses in the absence of membrane is helpful for
formulating hypotheses concerning mechanisms of membrane fusion (Bullough et
al., 1994; Rey et al., 1995; Fass et al., 1996; Chan et al., 1997; Weissenhorn et al.,
1997; Malashkevich et al., 1998; Weissenhorn et al., 1998; Baker et al., 1999; Kobe
et al., 1999; Malashkevich et al., 1999; Yang et al., 1999). However, these structures
might not be the same during the membrane fusion event, since conformational
changes of both the viral proteins and the membrane occur. Therefore, the stages
leading to the actual fusion event are mostly still unknown. In an effort to shed
light on this complex phenomenon, the interaction between model membranes and
synthetic peptides that mimic different regions within the envelope proteins of several viruses were studied. The synthetic peptide approach is supported by many
studies, some of which showed that non-covalently linked fragments of membrane
proteins are biologically active. These studies also revealed that transmembrane segments of membrane proteins can coassemble in a specific manner and that these
interactions can be used to predict the functional structure of membrane proteins
(reviewed in Lemmon and Engelman, 1994; Von Heijne, 1994; Shai, 1995).
The present review will focus on the identification of several functional regions
within the fusion proteins of several viruses, which are directly involved in conformational changes and the actual membrane fusion event. Peptides that can interfere
with the function of these regions have been shown to be potent inhibitors of viralcell fusion. Their plausible mode of action will also be discussed.
THE N-TERMINAL FUSION PEPTIDE DOMAIN
The binding of a fusion protein to its receptors induces a conformational change
in the glycoprotein that results in exposure of a previously hidden hydrophobic Nterminal domain, named ‘‘fusion peptide’’, and its penetration into the target cell
membrane. Fusion peptides from different viruses share high homology (Hsu et al.,
1981; Gallaher, 1987). Evidence for the role of the fusion peptide domain in
mediating membrane fusion comes from mutagenesis studies in intact envelope proteins of several viruses (Bosch et al., 1989; Freed et al., 1992; Delahunty et al., 1996;
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Pritsker et al., 1999), as well as from studies with synthetic fusion peptides (Lear
and DeGrado, 1987; Slepushkin et al., 1990; Burger et al., 1991; Clague et al., 1991;
Martin et al., 1991; Yeagle et al., 1991; Epand et al., 1994; Nieva et al., 1994;
Rapaport & Shai, 1994; Gray et al., 1996; Kliger et al., 1997; Ruiz-Arguello et al.,
1998; Peisajovich et al., 2000a). It has been shown that the ability of a particular
peptide to induce membrane fusion is highly dependent on its sequence, and most
mutations, even of a single amino acid, severely reduced or abolished its fusogenic
activity (Delahunty et al., 1996; Durell et al., 1997; Pécheur et al., 1999). This is
different from other families of membrane binding peptides such as antibacterial
peptides, pore forming cytolytic peptides and surfactants, which also exert their
activities by direct interaction with lipid constituents of the cell membrane, but
whose sequences can be altered significantly without affecting their biological function. This property might be important towards the development of antiviral drugs
based on the fusion peptides.
The significance of studying synthetic fusion peptides has been demonstrated
by several observations: (i) there is a direct correlation between the effects of
mutations in the intact protein and the peptide analogues (Freed et al., 1992; Horvath and Lamb, 1992; Rapaport and Shai, 1994; Pereira et al., 1995; Martin et al.,
1996; Kliger et al., 1997; Pritsker et al., 1999), (ii) the fusion activity of synthetic
peptides, measured in ûitro, is sensitive to factors (such as pH or the addition of
inhibitory agents) that affect the infectivity of the virus in ûiûo (Wharton et al., 1988;
Pereira et al., 1997), and (iii) synthetic fusion peptides showed anti-viral activity,
suggesting that they can accurately model and interact with functional domains of
the viral protein (Slepushkin et al., 1993; Kliger et al., 1997; Pritsker et al., 1999)
(see more details in the following paragraphs).
Although the detailed molecular mechanism of the actual fusion event is still
unknown, insertion of the fusion peptides into the cell (Harter et al., 1989; Stegmann
et al., 1989; Tsurudome et al., 1992; White, 1992; Pak et al., 1994), viral (Ruigrok
et al., 1988; Wharton et al., 1988), or both membranes (Guy et al., 1992; Hughson,
1995; Stegmann et al., 1995) has been postulated to induce local membrane dehydration (Zimmerberg et al., 1991) and to promote negative curvature in the bilayer
(Epand, 1998), factors that can help to overcome the energetic barriers associated
with the fusion process. Brasseur and co-workers (Brasseur et al., 1990; Brasseur,
1991) predicted that N-terminal fusion peptides can insert into the membrane with
an orientation oblique with respect to the water-membrane interface. This has been
shown experimentally for the N-terminal fusion peptides of Influenza (Tatulian et
al., 1995; Han et al., 1999), SIV (Epand et al., 1994; Martin et al., 1994; Colotto et
al., 1996; Bradshaw et al., 2000), HIV-1 (Martin et al., 1996; Kliger et al., 1997;
Pritsker et al., 1999), Bovine Leukemia virus (Voneche et al., 1992), and Sendai
virus (Rapaport and Shai, 1994; Ghosh and Shai, 1999). Oblique orientation should
increase the hydrophobic volume of the bilayer, thus promoting negative curvature
of the membrane (Epand et al., 1994), a factor known to facilitate fusion (Cheetham
et al., 1994; Chernomordik et al., 1995; Chernomordik et al., 1997; Epand, 1998).
In line of this, Richardson et al., (Richardson et al., 1980) have shown specific
inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino
acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides.
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Further studies revealed that such peptides are active only at a high concentration
and their activity is related to their ability to modify the membrane rather to interfere with the correct organization of the fusion proteins (Kelsey et al., 1990; Yeagle
et al., 1991; Yeagle et al., 1992; Epand et al., 1993; Stegmann, 1993). One of these
peptides, carbobenzoxy-D-phenylanine-L-phenylalanine-glycine (ZfFG), was studied
in detail in phospholipid membranes. It was suggested that it likely inhibits membrane fusion from the surface of the lipid bilayer, but not by forming a tight, stoichiometric complex with the phospholipids (Yeagle et al., 1992; Dentino et al.,
1995). In addition, Apolipoprotein A-I and its amphipathic helix peptide analogues
inhibit human immunodeficiency virus-induced syncytium formation probably via
interaction with the membrane (Owens et al., 1990). Further studies showed that a
22 amino acid-long fusion peptide inhibited HIV-1 envelope glycoprotein-induced
syncytium formation at concentrations of 1 µM (Slepushkin et al., 1993). The effect
of the peptide is certainly enhanced as its length increases, since it was found that
the 33 aa fusion peptide derived from the N-terminus of gp41 inhibited fusion at a
concentration of two orders of magnitude lower than that reported for the 22 amino
acid peptide. At peptide concentrations of 0.01 µM, the peptide caused 50% inhibition of fusion of HIV-1 envelope glycoprotein-expressing cells with human CD4
expressing cells, as revealed by fluorescence video imaging microscopy (Kliger et al.,
1997). A control peptide resembling the N-terminal modified version of the fusion
peptide of Sendai virus, was inactive in the inhibition assay (Kliger et al., 1997).
Recent studies have shown that fusion peptides specifically self-associate when
inserted into the membrane, and that the level of oligomerization is important for
their activity. This has been demonstrated with fusion peptides that adopt both an
α -helical secondary structure (Rapaport and Shai, 1994; Ghosh and Shai, 1999;
Ghosh et al., 2000) or a β -sheet structure (Pritsker et al., 1998; Pritsker et al., 1999;
Peisajovich et al., 2000a). This may explain the inhibitory effect of the 33-mer synthetic fusion peptide of HIV-1, since the peptide could assemble with the fusion
peptide domain of the intact protein and as a result interfered with its correct organization. Interestingly, an amino acid substitution that decreased the fusogenic
activity of fusion peptides in a model system decreased the size of the oligomers in
SDS-PAGE (Kliger et al., 1997; Pritsker et al., 1999). Similarly, such mutations also
decreased the activity of the intact virus or cells expressing the corresponding
mutated fusion proteins (Freed et al., 1992; Pritsker et al., 1999). For example,
mutation of the valine at position 2 in the amino terminal fusion peptide domain of
HIV-1 gp41 to glutamic acid resulted in an envelope glycoprotein that dominantly
interfered with both syncytium formation and infection mediated by the wild-type
HIV-1 envelope glycoprotein (Freed et al., 1992). This interference was not abolished
by excess of wild-type glycoprotein, suggesting that a higher-order envelope glycoprotein complex is involved in membrane fusion. The trans-dominant mutation also
inhibited HIV-1 envelope glycoprotein-mediated cell fusion when expressed in target
cells (Elson et al., 1994). In an attempt to understand this phenomenon, Kliger et
al. (Kliger et al., 1997) synthesized a 33-residue fusion peptide corresponding to the
V2E mutant. They showed that the mutant peptide is as active as the WT fusion
peptide in inhibiting HIV-1 envelope-mediated cell-cell fusion. The mutant peptide
Functional Domains within Fusion Proteins
539
could also self- and co-assemble with the WT fusion peptide in the membrane. Interestingly, the WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the
WT peptide, probably by forming inactive heteroaggregates. These data suggest that
specific interactions mediated by N-terminal fusion peptides are required to form
higher order oligomers necessary for membrane fusion (Kliger et al., 1997).
Attempts to define the oligomeric state of the HIV-1 envelope glycoprotein have
yielded conflicting results. Several reports indicated that the envelope glycoprotein
of HIV-1 is a tetramer in its membrane-bound state (Pinter et al., 1989; Schawaller
et al., 1989; Earl et al., 1990). On the other hand, other reports revealed that it
forms trimers (Weiss et al., 1990; Lu et al., 1995; Weissenhorn et al., 1996). In the
gp41 non-fusogenic form, the fusion peptide is buried, thus it is not responsible for
the oligomerization of the protein. However, once the envelope glycoproteins are
recruited to form a fusion complex, the fusion peptides will self-assemble. The size
of the aggregates formed by the WT fusion peptide of HIV-1 and its V2E and F11G
mutants was determined by using SDS as a membrane mimetic environment (Kliger
et al., 1997; Pritsker et al., 1999), as has been done with several other membrane
proteins (Lemmon et al., 1992; Simmerman et al., 1996). SDS-PAGE revealed that
the dominant form of the three peptides is a dimer, but that only the WT peptide
forms higher order oligomers, likely trimers兾tetramers. The inability of the V2E and
F11G mutants to form higher order oligomers may be correlated with their loss of
activity. Furthermore, the coassembly of the V2E with the WT peptide strengthens
the notion that the inhibition of membrane fusion exhibited by the WT and the V2E
mutant occurs via their association with the WT sequence of the intact protein. This
inhibition may occur because the V2E mutant decreases the portion of higher order
oligomers of WT, or because the V2E mutant forms non-functional high-order oligomers with the WT peptide. Since the V2E mutant gp41 elicits a dominant interfering
effect even in the presence of excess wild-type glycoprotein (Freed et al., 1992), the
second possibility is a more likely mechanism.
Interestingly, fusion peptide chirality was found not to be important for the
function of fusion peptides (Pritsker et al., 1998). Natural phospholipds are all Lforms and therefore provide the biological membrane interface with stereospecificity.
It has been shown that cell surfaces and phospholipid monolayers can stereospecifically recognize each other (Bohm et al., 1993; Hanein et al., 1994). In addition,
peptide chirality has been shown to be crucial for peptide-peptide interaction in
solution. Examples include the ribonuclease S-peptide兾S-protein complex (Corigliano et al., 1985), HIV-1 protease兾substrate complex (Milton et al., 1992) and the
coiled coil formed by the heptad repeats derived form the F1 fusion protein of
Sendai virus (Ghosh et al., 1998). Nevertheless, it was found that the WT fusion
peptide of HIV-1 gp41 and its enantiomeric (all D-amino acid) analogue bind equally
to phospholipid membranes and have equal potencies in inducing membrane fusion.
These findings indicate that the stereospecificity of the fusion peptide is not important for peptide-lipid interaction during the fusion process (Pritsker et al., 1998).
That peptide chirality is not a prerequisite for peptide-lipid interaction has been
shown in other cases as well. Enantiomers of amphipathic α -helical lytic peptides
such as the bee venom melittin and the antimicrobial peptides cecropin and
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Shai
magainin, possess lytic activity indistinguishable from that of the parent molecules
(Bessalle et al., 1990; Wade et al., 1990; Merrifield et al., 1995). These enantiomers
preserved the amphipatic α -helical structure of the wild type peptides, a structure
proposed to be prerequisite for their function. Since the biological function was
preserved, the enantiomeric peptides should be organized in the membrane similarly
to their parent all L-amino acid peptides. Similar results were obtained with all
D-amino acid Androctonin, a β -sheet antimicrobial peptide (Hetru et al., 2000).
Strikingly, it was demonstrated that the WT fusion peptide of HIV-1 gp41
associates with its enantiomeric wild-type fusion peptide in the membrane (Pritsker
et al., 1998). The D-amino acid peptide inhibits HIV-1 envelope-mediated cell–cell
fusion but does not inhibit cell–cell fusion mediated by HIV-2 envelope glycoprotein,
indicating that although chirality-independent, the interaction between fusion peptides within the membrane depends on their specific sequence.
THE CORE REGION OF FUSION PROTEINS
In addition to the fusion peptide domain, many fusion proteins also contain
one of more heptad repeat regions which are often adjacent to the fusion sequence
or to the transmembrane anchor domain (Gallaher et al., 1989; Chambers et al.,
1992). Site-directed mutagenic studies of HIV-1 gp41 and Measles virus F protein
indicate that heptad repeats found in these fusion proteins are required for membrane-fusion activity (Buckland et al., 1992; Dubay et al., 1992; Chen et al., 1993;
Doms et al., 1993; Reitter et al., 1995). Purified antibodies directed against a peptide
derived from a heptad repeat of gp41 effectively inhibit synctyia formation, thus
confirming the involvement of this segment in the membrane fusion process (Vanini
et al., 1993). These heptad repeats are speculated mainly to assist in the correct
folding of the proteins. The oligomeric structures of the heptad-repeat regions of
fusion proteins of Influenza (Carr and Kim, 1993), MoMuLV (Fass et al., 1996),
HIV (Wild et al., 1994a; Bernstein et al., 1995; Lu et al., 1995; Wild et al., 1995;
Chan et al., 1997; Weissenhorn et al., 1997), SIV (Blacklow et al., 1995) and simian
parainfluenza virus 5 fusion protein (SV5 F) (Baker et al., 1999) have been determined. Interestingly, similar core structures were found in a retrovirus such as HIV1 and paramyxoviruses such as SV5. Chan et al., (Chan et al., 1997) determined the
crystal structure of a complex composed of the N-terminal peptide, N36, and the Cterminal heptad repeat, C34. They found that three N36 helices form an interior,
parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel
manner into highly conserved, hydrophobic grooves on the surface of this trimer.
This structure shows striking similarity to the low pH-induced conformation of
influenza hemagglutinin. Baker et al., (Baker et al., 1999) have solved the crystal
structure of a fragment of SV5 F virus, revealing a 96 Å long coiled coil surrounded
by three antiparallel helices. This structure places the fusion and transmembrane
anchor of SV5 F in close proximity with a large intervening domain at the opposite
end of the coiled coil. Similar interactions were found between the N-terminal and
the C-terminal heptad repeats of Sendai virus (Ghosh et al., 1997; Ben-Efraim et
al., 1999). It is worth noting that the ectodomain portion of the paramyxovirus
transmembrane proteins is much longer than that of the corresponding segment in
Functional Domains within Fusion Proteins
541
e.g. HIV-1 (∼400 and ∼170 amino acid residues, respectively). Thus, the interaction
demonstrated between the heptad repeats of paramyxoviruses occurs between segments separated by ∼300 amino acid residues, while there are only ∼60 amino acid
residues between the heptad repeats of HIV-1.
Most interestingly, the core region has been found to be a target for antiviral
peptides. Synthetic peptides corresponding to heptad repeats adjacent to transmembrane domains of several viruses display selective antiviral activity for the virus of
origin, despite the fact that they are derived from ectodomains with different lengths.
This suggests that they can accurately model and interact with functional domains
of the viral protein. More specifically, peptides corresponding to the heptad repeats
of HIV-1 (Wild et al., 1992; Jiang et al., 1993; Wild et al., 1993), Sendai virus
(Rapaport et al., 1995; Ghosh et al., 1998), Respiratory Syncytial virus (Lambert et
al., 1996), Human Parainfluenza virus type 3 (Lambert et al., 1996), Measles virus
(Lambert et al., 1996), SV5 (Joshi et al., 1998) and Newcastle Disease virus (Young
et al., 1997; Young et al., 1999) have been shown to inhibit the fusion induced by
the respective virus, presumably by interfering with the conformational change
induced by the binding to the host-cell receptors. It has been speculated that they
bind to the N-terminal heptad repeats, before the C-terminal heptad repeats pack
into the grooves formed by the coiled coil. In this way, folding into the putative
fusion-active confirmation is prevented. For example, a synthetic peptide overlapping the C-terminal amphipathic helical segment of gp41 and its tryptophan-rich
sequence (Salzwedel et al., 1999) (DP178, amino acids 638–673) was reported to
inhibit virus infection at extremely low concentrations (Wild et al., 1994b). Remarkably, DP178 blocks cell fusion and viral entry at a concentration of less than 2 ng兾
ml in ûitro. Moreover, DP178 was reported to be a new, promising drug for treating
HIV-1 infected humans (Kilby et al., 1998). Recently, Weiss and colleagues demonstrated that DP178 binds gp41 and inhibits envelope-mediated membrane fusion,
but only after gp120 interacts with cellular receptors (Furuta et al., 1998). In another
recent study (Kliger and Shai, 2000), it was found that whereas DP178 perturbs
the partial α -helix nature of peptides corresponding to the leucine兾isoleucine zipper
sequence (N36 or N51), it cannot perturb the coiled-coil conformation, modeled by
the complex of N36 or N51 with C34. Therefore, it was suggested that the already
formed heterotrimer coiled-coil is not the target of inhibition by DP178. The later
results are consistent with a model in which DP178 acquires its inhibitory activity
by binding to an earlier intermediate of gp41, in which the N and C peptide regions
are not yet associated, thus allowing DP178 to bind to the leucine兾isoleucine zipper
sequence and consequently to inhibit transition to the fusion-active conformation
(Chan & Kim, 1998; Weissenhorn et al., 1999; Kliger and Shai, 2000). This model
is supported by the results of Blumenthal and co-workers, suggesting that the prehairpin intermediate stage appears to be induced rapidly upon interaction of gp120
with CD4 and the coreceptor, but is then relatively stable for several minutes (Jones
et al., 1998; Muñoz-Barroso et al., 1998), allowing DP178 to interact with the
exposed leucine兾isoleucine zipper sequence. Recently, Kim and co-workers designed
a peptide, IQN17, which properly presents a prominent pocket on the surface of the
central trimeric coiled coil within gp41 (Eckert et al., 1999). Utilizing IQN17 and
mirror-image phage display, they identified cyclic, D-peptide inhibitors of HIV-1
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Shai
infection that share a sequence motif. A 1.5 Å cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these
inhibitors make intimate contact with the gp41 pocket.
Blumenthal and his colleagues (Muñoz-Barroso et al., 1998) developed a new
three-color assay to keep track of the cell into which fluorecent lipids and兾or solutes
are redistributed. Lipid and solute redistribution occur as a result of opening a lipidpermissive fusion pore and a solute-permissive fusion pore, respectively. They found
that DP178 completely inhibited solute redistribution at 50 ng兾ml, but not lipid
redistribution. This difference was maintained up to 6 h of coculture of gp120–41
expressing cells with target cells, indicating that DPA178 can ‘‘clamp’’ the fusion
complex in the lipid mixing intermediate for very long time periods. On the other
hand the heptad repeat derived from the N-terminal domain was less potent, but
with no differences between lipid and aqueous dye redistribution at the different
inhibitor concentrations. Interestingly, an opposite trend was found with the heptad
repeats of SV5 F protein of paramyxovirus (Joshi et al., 1998). The C-terminal heptad repeat was found to be a potent inhibitor of both the lipid mixing and the
aqueous content mixing fusion activity of the SV5 F protein, whereas the N-terminal
heptad repeat inhibited cytoplasmic content mixing but not lipid mixing, leading to
a stable hemifusion state. Recent studies done by Shai and his colleagues (Kliger
et al., 2000), reviewed in (Dimitrov, 2000)) showed that a shifted C-terminal heptad
repeat derived from gp41 of HIV1 (termed C34 (Chan et al., 1997)) is a potent
inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of
HIV-1, similarly to what has been found in the cases of SV5, thus strengthening the
similarities between retro- and parmyxoviruses. In the same study, structural and
functional characterization of both C34 and DP178 suggested that DP178 has a
second binding site, likely its corresponding region within the intact gp41 within the
membrane milieu (Kliger et al., 2000, reviewed in Dimitrov, 2000).
It is interesting to note that DP-107, a synthetic peptide corresponding to the
N-terminal heptad repeat of HIV-1 is 1000 times less active than DP-178. Similarly
to this result, it was found that the N-terminal heptad repeat of Sendai virus is much
less active than its C-terminal heptad repeat (Ghosh and Shai, 1999), thus further
supporting the notion that retro- and paramyxoviruses share common structural
motifs. Interestingly, however, attachment of the N-terminal fusion peptide to the
N-terminal heptad repeat of Sendai virus significantly enhanced its inhibitory
activity (Ghosh and Shai, 1999).
Studies on the mode of inhibition of the C-terminal heptad repeat of Sendai
virus, SV-465, revealed that only the wild type peptide was active, but not its derivatives, one with two heptadic leucines substituted with two alanines, and the second
an antiomeric (all D-amino acids) version of SV-465 (Ghosh et al., 1998). This was
consistent with the finding that only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein, namely, the
N-terminal heptad repeat and a leucine zipper-like region located in the middle of
Sendai virus F1 protein. The finding that SV0-465 inhibited equally when added to
virions before or after their attachment to cells, and that its inhibitory effect was
not time dependent, suggests that SV-465 can disturb a step which occurs after the
binding of virions to the target cells.
Functional Domains within Fusion Proteins
543
A LEUCINE ZIPPER REGION BETWEEN THE N- AND C-TERMINAL
HEPTAD REPEATS OF PARAMYXOVIRUSES
Paramyxoviruses contain an additional leucine zipper motif located in between
the N- and C-heptad repeats within the F1 fusion protein. The leucine zipper region,
designated SV-269 (amino acids 269-107), in the ectodomain of the Sendai virus
protein is extremely conserved in the family of paramyxoviruses (Ghosh et al., 1997).
Furthermore, most of the heptadic leucines兾isoleucines are conserved in other
members of paramyxoviruses, suggesting a similar role in the corresponding fusion
proteins. To find a role for this region in Sendai virus mediated red blood cell hemolysis, Ghosh et al., (Ghosh et al., 1997) synthesized SV-269, a 39 amino acid long
peptide and fluorescently labeled it. A mutant peptide, MuSV-269, with only two
amino acids (one heptadic leucine and a glutamic acid) interchanged in their positions was also synthesized and investigated. Functional studies revealed that the
wild type leucine zipper motif, SV-269, is a potent inhibitor of Sendai virus mediated
cell fusion. It was also found that SV-269 exists in an oligomeric state in solution
whereas the mutant peptide, MuSV-269, does not. Interestingly, it was found that
SV-269, but not MuSV-269, coassembled in solution with the N- and C-terminus
heptad repeats, however, only with their longer versions that showed antiviral
activity. That SV-269 can self- and coassemble with other domains of the Sendai
virus fusion protein is supported by the finding that the peptide binds specifically to
Sendai virions whereas its mutant MuSV269 did not (Ghosh et al., 1997).
Lamb and co-workers have studied this central leucine zipper (amino acids 255293) in the F protein of the paramyxovirus SV5 (Dutch et al., 1999). They found
that whereas the N- and C-terminal heptad repeats interact forming a thermostable,
alpha-helical trimer of heterodimers, the central leucine zipper interacts also with
the C-terminal heptad repeat forming an helical complex; however, this complex is
not thermostable. Furthermore, the peptide 255-293 was not able to destabilize the
complex formed by the N- and C-terminal heptad repeats.
The role of the central leuzine zipper between amino acids 268 and 289 in the
structure and function of the Newcastle disease virus (NDV) F protein was explored
by Morrison and co-workers (Sergel et al., 2000) by introducing single point
mutations into the F gene cDNA. The mutations affected either folding of the protein or its fusion activity. Two mutations, L275A and L282A, likely interfered with
folding of the molecule since these proteins were not proteolytically cleaved, were
minimally expressed at the cell surface, and formed aggregates. They found that the
L268A protein was fusion inactive in the presence or absence of HN protein
expression. However, very surprisingly, the mutant L289A protein mediated syncytium formation in the absence of HN protein expression although the HN protein
enhanced the fusion activity. These results show that a single amino acid change in
the F(1) portion of the NDV F protein can alter the stringent requirement for HN
protein expression in syncytium formation. Similar results were obtained by Ito
et al., (Ito et al., 2000). The authors analyzed chimeras of HN-dependent and HNindependent SV5 F proteins and showed that, in addition to Pro 22 at the F2 Nterminus, Glu1 31 (located in the N-terminal heptad repeat) and Ala 290 (located in
the central heptad repeat) are important for the HN-independent fusion activity of
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the SV5 F protein. This suggests a possible role for these regions in the interaction
of the F and HN proteins.
MEMBRANE-INDUCED CONFORMATIONAL CHANGES OF THE
FUSION PROTEIN DURING THE FUSION PROCESS
Several conformational changes characterize the fate of viral fusion proteins.
The posttranslational cleavage results in a conformational change manifested by an
increase in the exposed hydrophobicity, which is thought to be related with the
exposure of the fusion peptide (Skehel et al., 1982). Binding to the host-cell receptor
induces a second change in conformation that is believed to include the insertion of
the fusion peptide into the target membrane and the packing of the C-terminal
heptad repeats against the grooves of the coiled-coil formed by the N-terminal heptad repeats, resulting in the structure observed by X-ray crystallography or NMR
(Bullough et al., 1994; Fass et al., 1996; Chan et al., 1997; Weissenhorn et al., 1997;
Malashkevich et al., 1998; Weissenhorn et al., 1998; Baker et al., 1999; Kobe et al.,
1999; Malashkevich et al., 1999; Yang et al., 1999). Using electron paramagnetic
resonance analysis, Shin and co-workers (Yu et al., 1994; Rabenstein and Shin,
1995) showed that cysteine-substituted peptides comprising the loop region and part
of the N-terminal heptad repeat of Influenza virus fusion protein (HA) and the Nterminal heptad repeat of HIV-1 fusion protein (gp41) insert reversibly into phospholipid vesicles. Based on these results, the authors suggested that binding of the
N-terminal heptad-repeat regions to the membrane could bring the viral and cell
membranes closer together and facilitate fusion. Ben-Efraim et al. (Ben-Efraim et
al., 1999) analyzed the interaction between peptides corresponding to the N- and Cterminal heptad repeats from Sendia virus fusion protein, in the presence and in the
absence of membranes. They showed that in aqueous environment, the peptides coassemble into an α -helical complex, presumably with a structure similar to that
of the core complex of the homologous SV5 fusion protein determined by x-ray
crystallography (Baker et al., 1999) and other fusion proteins (Bullough et al., 1994;
Fass et al., 1996; Chang et al., 1997; Weissenhorn et al., 1997; Malashkevich et al.,
1998; Weissenhorn et al., 1998; Kobe et al., 1999; Malashkevich et al., 1999; Young
et al., 1999). However, in the presence of phospholipid membranes, this complex
binds strongly to the membrane’s surface and dissociates therein. This led Shai and
co-workers to suggest an ‘‘umbrella’’ like model for the unfolding of the F1 protein
(Figure 1 upper panel). In agreement with these observations, NMR studies showed
that the N-terminal repeat of the homologous Newcastle Disease virus protein has
an α -helical structure in SDS, consistent with the idea that it binds parallel to the
bilayer, as a monomer, with its hydrophobic face buried in the membrane (Young
et al., 1999). Furthermore, Shai and co-workers (Kliger et al., 2000b) found that
recombinant proteins corresponding to the ectodomain of HIV-1 fusion protein,
but lacking the fusion peptide, bind membranes and consequently undergo a major
conformational change. As a result, the protease-resistant core becomes susceptible
to proteolytic digestion. Accordingly, they showed that synthetic peptides corresponding to the segments that construct this core oligomerize in aqueous solution,
Functional Domains within Fusion Proteins
545
Fig. 1. The ‘‘Umbrella’’ model for the actiûation of the fusion protein of Sendai ûirus.
Upper panel: Model without the internal fusion peptide. Binding of the viral surface glycoprotein
to cell receptors (Fig. 1, step a) results in a conformational change of the fusion protein, which
leads to the formation of a trimeric coiled-coil (Fig. 1, step b). This leads to the exposure and
insertion of the N-terminal fusion peptide in the target membrane (Fig. 1, step b), following by
dissociation of the core region upon binding to membranes (Fig. 1, step c).
Lower Panel: A revised model that includes the internal fusion peptide. The conformational
changes due to receptor binding expose both the internal and the N-terminal fusion peptides.
Either the N-terminal fusion peptide (Fig. 1, step d) or the internal fusion peptide (Fig. 1, step
e) insert into the target membrane first, followed by binding of both the N-terminal and the Cterminal heptad repeats to the membrane. This causes the ‘‘umbrella’’-like opening of the coiledcoil Fig. 1, step f). The membrane-induced conformational change consequently causes the cellular and viral membranes to approach each other. Subsequently, both the N-terminal and the
internal fusion peptides induce the actual merging of the membranes. The figure shows also
potential targets for the peptides inhibitors.
but dissociate upon binding to the membrane. This suggests that both the N-terminal
and C-terminal heptad-repeats can assist in bringing the viral and cellular membranes closer, facilitating the subsequent merging. The similarity between what was
found in HIV-1, a retrovirus, and in Sendai, a paramyxovirus, suggests that these
distantly related viruses share common steps in their fusion mechanism.
Studies on the mechanism of inhibition of viral infection by a synthetic peptide
(DP-178 or T-20 modeled after a membrane-proximal region located downstream of
the C-terminal heptad repeat of HIV-1 gp41, prompted Kliger et al., (Kliger et al.,
546
Shai
2000a) to postulate a further conformational change for HIV-1 gp41. They showed
that DP178 could block two steps in gp41 conformational cascade at different affinities. The low affinity site represents inhibition of host-cell receptor-induced conformational change, before the coiled coil binds to the membrane (Kliger and Shai,
2000). The high affinity site represents inhibition of the newly postulated change in
the oligomerization state of gp41: DP-178 interacts with its corresponding segment
in the full-length protein, and thus inhibits the recruitment of several gp41-membrane complexes which leads to fusion pore formation (Kliger et al., 2000a).
A SECOND FUSION PEPTIDE IN THE FUSION PROTEINS OF
PARAMYXOVIRUSES
Orthomyxo- and retrovirus fusion proteins are thought to contain only one
N-terminal fusion peptide (Gallaher, 1987; White, 1990), whereas paramyxoviruses
contain, in addition to the N-terminal fusion peptide, a second one in the interior
of the fusion protein (Peisajovich et al., 2000b). More specifically, very recently,
Peisajovich et al. (Peisajovich et al., 2000b) searched for regions other than the Nterminal fusion peptide in the Sendai virus F protein, that could participate in the
actual merging of the viral and cellular membranes. The rationale was that the extracellular domain of the fusion protein of Sendai virus consists of more than 560
amino acids (Homma and Ohuchi, 1973; Scheid and Choppin, 1977), and therefore
it is unlikely that the whole membrane fusion process is accounted for only by a
small N-terminal fusion peptide. Strikingly, they found a region corresponding to
amino acids 214-226 highly homologous to the fusion peptide of the HIV-1 and
RSV envelope glycoproteins. Synthetic peptides corresponding to this region, as well
as to the elongated version of the peptide, were able to induce membrane fusion of
large unilamellar vesicles to a substantially higher extent than the N-terminal 33amino acid fusion peptide (Peisajovich et al., 2000b). Furthermore, an homologous
peptide from Measles virus F1 protein was also synthesized and found to be active,
suggesting that this novel finding is a common feature of paramyxoviruses (Samuel
O., and Shai, Y., unpublished results). These findings led Shai and co-workers to
postulate a revised model for paramyxovirus-induced membrane fusion (Figure 1,
lower panels). According to this model, the conformational changes due to receptor
binding expose both the internal and the N-terminal fusion peptides, Although we
can not rule out the possibility that the N-terminal fusion peptide inserts into the
target membrane first (Fig. 1, step d), the existence of a second fusion peptide consecutive to the N-terminal heptad repeat, located on top of the coiled coil in this
conformation, opens the possibility that the initial interaction with the target membrane is achieved by means of the internal fusion peptide, which oligomerizes and
adopts an α -helical structure upon membrane binding, as suggested in Fig. 1, step
e. In this conformation, the N-terminal fusion peptide might be embedded in the
viral membrane, in agreement with the models suggested by (Kozlov and Chernomordik, 1998; Bentz, 2000). The affinity of both the N-terminal and the C-terminal
heptad repeats to the membrane causes the ‘‘umbrella’’-like opening of the coiledcoil (Ben-Efraim et al., 1999) Fig. 1, step f). This membrane-induced conformational
change consequently causes the cellular and viral membranes to approach each
Functional Domains within Fusion Proteins
547
other. Subsequently, both the N-terminal and the internal fusion peptides induce the
actual merging of the membranes. Obviously, further experiments are needed to
determine whether both fusion-active segments destabilize the same membrane. The
location of the internal and the N-terminal fusion peptides, both in the same membrane (Fig. 1, step f) is therefore speculative.
The importance of the second fusion peptide in Sendai virus-induced membrane
fusion was highlighted by the finding that a synthetic peptide corresponding to this
fusogenic region, namely SV-201, inhibits the fusion between the virus and red blood
cells (Ghosh and Shai, 1998). Ghosh and Shai (Ghosh et al., 1998; Ghosh and Shai,
1998) found that SV-201 specifically inhibited virus-mediated hemolysis only when
added to virions prior to their attachment to red blood cells. Sendai virus-mediated
hemagglutinin assay in the presence of SV-201 demonstrated that the peptide does
not disturb the binding of virions to the target red blood cells. Mutant peptides were
either inactive or only slightly active. In an attempt to understand the role of SV201 in membrane fusion and its mechanism of inhibition, SV-201 and its shorter
and longer reversion, as well as several mutants were synthesized and investigated
for their inhibitory effect, their fusogenic activity, their structure in the membrane
bound state, and their ability to oligomerize in solution and when bound to membranes (Ghosh et al., 2000). The results helped to understand SV-201 mechanism
of inhibition and strongly support the ‘‘umbrella’’ model of Sendai-virus induced
membrane fusion (Peisajovich et al., 2000b).
The differences in the inhibitory activity, their ability to bind to Sendai virions,
and the oligomerization state of these peptides in aqueous solution and within the
membrane was used to distinguish whether SV-201 inhibits the entry of Sendai virus
into the host cell by binding to its counterpart in the F1 protein before or after the
internal fusion peptide binds to the membrane. The data revealed that both SV-201
and its inactive short version were able to oligomerize in the membrane, whereas
only SV-201 oligomerized in aqueous solution (Ghosh and Shai, 1998; Ghosh et al.,
2000; Peisajovich et al., 2000b). Furthermore, only SV-201 interacts in a specific
manner with active virions in the absence of target cells, whereas this interaction
was significantly reduced after incubation of the virus at 60°C, which results in a
conformational change for the F protein (Wharton et al., 2000). If inhibition is
accomplished by binding to its corresponding region in the membrane, it would be
expected that both peptides are inhibitors. On the other hand, if inhibition is
achieved by binding to the corresponding region in aqueous solution, it would be
expected that only SV-201 will be an inhibitor. Since SV-201 but not its shorter
version inhibits viral entry to RBC, Shai and co-workers postulated that SV-201
binds to its counterpart in the F1 protein before the internal fusion peptide binds to
the membrane, thus blocking the receptor-induced conformational change, as
depicted in Figure 1. It should be noted that although SV–201 oligomerizes under
the conditions used in the inhibition experiment, the oligomerization process is
reversible (Ghosh and Shai, 1998); therefore part of the peptides are available to
interact with their counterparts in the F1 protein.
In summary, several functional domains within fusion proteins from different
viral families are involved in conformational changes after receptor binding and in
the membrane fusion event. These studies suggest an ‘‘umbrella’’ like model for the
548
Shai
Fig. 2. The ‘‘Umbrella’’ model for HIV-1-induced membrane fusion: (a) Native state
of HIV-1 envelope glycoprotein. (b) Upon binding of gp120 to cellular receptors, a
conformational change in gp41 is induced, allowing an extension of the N-terminal
fusion peptide toward the cellular membrane. (c) Subsequently, the leucine兾isoleucine
zipper and the C-terminal amphipathic sequences are assembled into a core domain
which is stable to enzymatic degradation (see inset SDS PAGE). (d) The affinity of both
segments to the membrane causes the opening of this three-hairpin structure. The leucine兾isoleucine zipper and the C-terminal amphipathic sequences bind to the cellularbound core is susceptible to enzymatic degradation (see inset SDS PAGE). Further
steps, not all of them defined, are needed for complete fusion. The figure shows also
potential targets for the peptides inhibitors inhibitors.
membrane induced conformational change of fusion proteins. Peptides resembling
these regions have been shown to have specific antiviral activity, presumably because
they interfere with the corresponding domains within the viruses (Figure 2, dashed
boxes). Studies along this line shed light into the molecular mechanism of membrane
fusion induced by envelope glycoproteins and suggest that fusion proteins from different viral families share common structural and functional motifs.
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