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Journal of Virology, May 2001, p. 4268-4275, Vol. 75, No. 9
Institute of Virology, University of Vienna,
A-1095 Vienna, Austria
Received 28 September 2000/Accepted 29 January 2001
The envelope protein E of the flavivirus tick-borne encephalitis
(TBE) virus promotes cell entry by inducing fusion of the viral
membrane with an intracellular membrane after uptake by endocytosis.
This protein differs from other well-studied viral and cellular fusion
proteins because of its distinct molecular architecture and apparent
lack of involvement of coiled coils in the low-pH-induced structural
transitions that lead to fusion. A highly conserved loop (the cd loop),
which resides at the distal tip of each subunit and is mostly buried in
the subunit interface of the native E homodimer at neutral pH, has been
hypothesized to function as an internal fusion peptide at low pH, but
this has not yet been shown experimentally. It was predicted by
examination of the X-ray crystal structure of the TBE virus E protein
(F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a
specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and
related properties. Replacement of Leu with hydrophilic amino acids
strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a
Phe mutant still retained a significant degree of fusion activity.
Liposome coflotation experiments showed that the fusion-negative Asp
mutant did not form a stable interaction with membranes at low pH,
although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target
membranes during fusion.
Enveloped viruses enter cells by
fusing their membranes with a host cell membrane, either at the cell
surface or at an internal site after uptake by endocytosis. This is
mediated by metastable surface proteins that undergo a triggered
conformational change upon binding to a receptor or exposure to the
acidic environment of the endosome, allowing a previously buried
portion of the protein, the fusion peptide, to insert into the target
membrane (22). Exposure and insertion of the fusion
peptide is believed to be the crucial step in the initiation of the
fusion process, although further conformational changes may be required
for achieving the complete merger of the lipid bilayers.
One structurally related group of well-characterized viral fusion
proteins includes the spike proteins of influenza A and C viruses,
human immunodeficiency virus and other retroviruses, paramyxoviruses,
and filoviruses such as Ebola virus (for reviews, see references
44, 48, and 5). These fusion proteins require proteolytic
cleavage for activity (27), and their fusion peptides reside at or near the N-terminal end of the membrane-anchored subunit.
When activated by the appropriate trigger, they all adopt a
characteristic six-helix rod-like structure with a long coiled coil at
the trimer interface. During the formation of the core, the fusion
peptide is translocated to the tip of the rod, permitting it to
interact with the target membrane and initiate fusion. These observations have led to a general model for virus-induced fusion, which seems to apply also to fusion by some cellular proteins such as
SNARES (24, 44, 48).
In contrast, the fusion proteins of several other enveloped viruses
(e.g., rhabdoviruses, alphaviruses, and flaviviruses) appear to be
different. They are not proteolytically cleaved during maturation, they
are not predicted to form coiled coils (43), and they
appear to use internal sequences (15, 21, 22, 25) rather
than an N-terminal or N-proximal fusion peptide to mediate fusion. The
only protein of this class for which a high-resolution structure is
currently available is the envelope glycoprotein E of the flavivirus
tick-borne encephalitis (TBE) virus (36). The striking
lack of structural similarity between the native forms of the fusion
proteins of flaviviruses and orthomyxoviruses (41, 49)
suggests that they might use fundamentally different mechanisms to
carry out essentially identical functions.
The flaviviruses are small enveloped viruses that are responsible for a
number of mosquito- and tick-borne diseases such as yellow fever,
dengue fever, Japanese encephalitis, West Nile encephalitis, and
tick-borne encephalitis (31). The interior of the virion consists of an isometric nucleocapsid containing the unsegmented positive-stranded RNA genome complexed with the capsid protein C. The
outer surface contains two membrane-anchored proteins: the envelope
glycoprotein (E), which mediates fusion in the endosomal compartment
after endocytosis, and the small membrane protein M. The E proteins of
all mosquito- and tick-borne flaviviruses have at least 40% amino acid
identity and their six intramolecular disulfide bridges are conserved,
indicating a common overall structure (36). Flaviviruses
are synthesized intracellularly as immature particles containing a
larger precursor form of the M protein (prM), which is subsequently
cleaved by a cellular protease to yield the mature virion
(37).
The X-ray crystal structure of the E protein of TBE virus at
2-Å resolution (36) revealed that it is an
elongated head-to-tail homodimer that, rather than forming a spike,
lies parallel to the surface of the virion, anchored in the membrane at
its distal ends (Fig. 1A). The external
portion of each subunit consists of three structural domains (I, II,
and III), which correspond to previously defined antigenic domains
(29). Cryoelectron microscopy studies with
recombinant subviral particles (RSPs) have shown that there are
specific lateral interactions between neighboring E dimers
(13). This results in an icosahedral lattice structure composed of local threefold assemblies of E dimers (Fig. 1B). Exposure
to acidic pH induces a conformational change that weakens the subunit
interactions within the dimer while strengthening lateral interactions
with other E proteins, presumably those in adjacent positions, giving
rise to a homotrimeric form of E (2, 46). The exposure of
the fusion peptide and binding to the target membrane probably occurs
during this reorganization of the viral envelope structure. Fusion then
proceeds rapidly without any requirement for specific proteins or
lipids in the target membrane (8).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4268-4275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutational Evidence for an Internal Fusion Peptide
in Flavivirus Envelope Protein E
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (37K):
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FIG. 1.
(A) Ribbon diagram of the ectodomain portion of the TBE
virus E protein dimer (36) viewed from the top
(perpendicular to the virion surface), with the individual subunits
colored red and blue. The C-terminal portion of the protein, whose
structure is not known, extends downward from domain III at each end of
the dimer to anchor the protein in the viral membrane. Domains I, II,
and III are labeled by roman numerals, and the cd loop at the tip of
domain II is colored yellow. The position of Leu 107 is shown by a
green ball. (B) Schematic representation of a threefold assembly of E
dimers as determined by cryoelectron microscopy and icosahedral
reconstruction of RSPs (13). Domains are indicated by
roman numerals, and filled yellow circles represent the cd loop of
domain II. (C) Zoom of the top view in panel A, showing details of the
cd loop and vicinity. Amino acids 100 to 108 (the cd loop) as well as
Cys 74 are shown as ball-and-stick representations and colored by atom
(green, C; blue, N; red, O; yellow, S). The first
N-acetylglucosamine residue of the N-linked glycan, which
covers the cd loop but is attached to Asn 154 of the other subunit, is
shown in gray. Other amino acids are represented as sticks.
It was proposed several years ago based on indirect evidence that the sequence element containing amino acids 98 to 110 might serve as an internal fusion peptide (39, 40). This region is highly conserved among flaviviruses, with the exception of position 104, which is occupied by histidine in tick-borne and glycine in mosquito-borne flaviviruses. The X-ray crystal structure (36) showed that this conserved region lies at the distal tip of each subunit (Fig. 1A). Residues 100 to 108 constitute a loop (cd loop) between the antiparallel strands c and d of domain II, which is buried in a hydrophobic pocket formed at the interface between domains I and III of the other monomer. In the native TBE virus E protein dimer, it is also covered by the N-linked oligosaccharide from its partner subunit. This region is highly constrained by multiple interactions, including several internal hydrogen bonds, one salt bridge (Asp 98 and Lys 110), and one disulfide bond (Cys 74 and Cys 105) that connects the cd loop to the bc loop of the same domain (Fig. 1C).
Using the X-ray crystal structure of the TBE virus E protein as a basis for rational mutagenesis, we have attempted to obtain direct experimental evidence for the involvement of this sequence in fusion. As a model system for these studies, we used noninfectious RSPs, which are icosahedrally symmetrical structures composed only of the mature viral surface proteins embedded in a lipid membrane and lacking the nucleocapsid and RNA genome (3, 13, 42). They are assembled intracellularly in cells expressing the prM and E proteins and undergo a maturation process similar to that of the whole virion, including glycosylation, transport through the Golgi complex, furin-mediated cleavage of prM, and secretion (3, 42). At mildly acidic pH, RSPs are capable of inducing cell-cell fusion (42) as well as fusion with artificial membranes (8). In the latter case it was shown that RSPs fuse with the same pH dependence, kinetics, and lipid dependence as whole virions.
Using the known structure of the TBE virus E protein and taking into consideration the structural constraints within the cd loop region, Leu 107 was identified as a target for single amino acid substitutions that could be introduced without interfering with the interactions required for dimer and particle formation. The functional analysis of RSPs containing mutations at position 107 provides evidence that the cd loop might be directly involved in membrane interactions during fusion.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of mutants. Recombinant plasmids containing substitutions at codon 107 of the E gene were derived from the wild-type plasmid SV- PEwt (1), a cDNA clone containing the prM and E genes of TBE virus strain Neudoerfl (GenBank accession no. U27495) under the control of the simian virus 40 early promoter, which yields secreted RSPs when expressed in COS cells (3). Mutant plasmids were constructed by replacing a 499-bp AgeI-BpuI fragment (nucleotides 960 to 1459) from SV-PEwt with a 196-bp PCR-generated Sau3AI-BpuI fragment containing the desired mutation and a 303-bp AgeI-Sau3AI fragment from plasmid SV-E07 (1), which is identical to the corresponding region of SV-PEwt except for a silent mutation at nucleotide 1257 that facilitated cloning by eliminating an additional Sau3AI site. The mutated codons at position 107 (TTC for Phe, GAC for Asp, and ACG for Thr) were included in the PCR primers at nucleotides 1291 to 1293. The wild-type and mutant plasmids were propagated in Escherichia coli strain HB101, purified using a Qiagen plasmid mega kit, and sequenced throughout the prM and E coding regions to confirm that only the desired mutations were present.
Production of RSPs. COS-1 cells were transfected with recombinant plasmids by electroporation as described previously (4). RSPs were harvested by pelleting from cell supernatants 48 h after transfection and purified on sucrose gradients (42). For membrane fusion assays, the particles were metabolically labeled with 1-pyrenehexadecanoic acid (Molecular Probes, Leiden, The Netherlands) as described by Corver et al. (8).
Electron microscopy. RSPs were placed onto glow-discharged Formvar-carbon-coated cupron grids (Agar Scientific, Stansted Essex, England) and allowed to adsorb for 5 min. Samples were stained for 4 min with 1% uranyl acetate (pH 4.5) and viewed using a Zeiss EM10 electron microscope at a magnification of ×50,000.
Membrane fusion assay. Fusion of pyrene-labeled RSPs with liposomes was measured by monitoring the decrease in pyrene excimer fluorescence at 480 nm with excitation at 343 nm as described by Corver et al. (8). Briefly, RSPs were mixed with liposomes (total phospholipid, 0.2 mM) consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio, 1:1:1:1.5) in a continuously stirred fluorimeter cuvette at 37°C (final volume, 0.5 ml). Fluorescence was monitored continuously using a Perkin-Elmer LS-50B9 fluorescence spectrophotometer. The fusion reaction was initiated by the addition of 300 mM 2-(N-morpholino)ethanesulfonic acid (MES) to yield a final pH of 5.5. The degree of fusion was calculated taking the initial excimer fluorescence after mixing to represent 0% fusion and the fluorescence after dispersion of the RSP-liposome mixture with the detergent octa(ethylene glycol)-n-dodecyl monoether (Fluka, Buchs, Switzerland) to represent 100% fusion.
HA assay. Hemagglutination (HA) of goose erythrocytes was carried out at pH 6.4 by the method of Clarke and Casals (7).
Analysis of conformational change. RSP preparations (E protein concentration, 5 µg/ml) in TAN buffer (0.05 M triethanolamine [pH 8.0], 0.1 M NaCl) plus 0.1% bovine serum albumin were acidified by the addition of a 0.05 M Tris-maleate buffer or 0.05 M MES containing 0.1 M NaCl and 0.1% bovine serum albumin to yield the desired pH. After a 10-min incubation at 37°C, the pH was adjusted to 8.0.
The conformational state of the E protein was assessed in a four-layer enzyme-linked immunosorbent assay (ELISA) using a TBE virus-specific guinea pig antiserum as catching antibody and mouse monoclonal antibodies (MAbs) for detection (20). The pattern of reactivity of the E protein with a panel of 18 conformation-sensitive MAbs was determined as described previously (42).Sedimentation analysis. The conversion of E dimers to trimers at low pH was measured by sedimentation analysis as described previously (2, 42). Samples were acidified and back-neutralized as described above and then solubilized for 1 h at room temperature with 0.5% Triton X-100. This material was then applied to a 7 to 20% (wt/wt) sucrose gradient made with TAN buffer and 0.1% Triton X-100 and centrifuged for 20 h at 38,000 rpm in a Beckman SW40 rotor at 15°C. Fractions (0.6 ml) were collected by upward displacement, and E protein was quantitated by four-layer ELISA after a 30-min denaturation with 0.2% sodium dodecyl sulfate at 65°C (19).
Liposome coflotation assay. RSPs were mixed with liposomes of the same composition used in the fusion assay (final phospholipid concentration, 6 mM) in a buffer consisting of 10 mM triethanolamine (pH 8.0) and 140 mM NaCl. After a 10-min incubation at 37°C, the mixture was acidified by the addition of 150 mM MES to yield a pH of 5.5 and incubated for another 10 min. The mixture was then back-neutralized by the addition of 150 mM triethanolamine to yield a final pH of 7.8 and mixed with sucrose to a final concentration of 20% (wt/wt). The 20% solution containing the sample (0.75 ml) was layered onto a 1-ml cushion of 50% sucrose in TAN buffer, after which two more sucrose layers (15%, 1.25 ml; 5%, 1 ml) were applied. The step gradients were centrifuged for 2 h at 4°C in a Beckman SW55 rotor at 50,000 rpm, and fractions (0.2 ml) were collected by upward displacement. E protein was quantitated by four-layer ELISA after treatment with sodium dodecyl sulfate (19).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mutagenesis of Leu 107. To investigate the possible involvement of the cd loop (Fig. 1) in membrane fusion, we made mutant RSPs and investigated their fusion-related properties. Our goal was to introduce amino acid substitutions that would lead to functional changes but would not disrupt dimer interactions or impair particle formation, maturation, and secretion. Close examination, however, revealed that most of the nonglycine residues of the cd loop region are involved in an extensive network of interactions that stabilize either the cd loop structure itself or its interactions with the other subunit of the dimer (36). One exception is Leu 107, whose side chain is on the exterior of the molecule and oriented away from the dimer interface (Fig. 1C), suggesting that amino acid substitutions at this position might be well tolerated.
Leu 107, like most of the cd loop region, is almost completely conserved among flaviviruses, but there are a few exceptions: the tick-borne Powassan virus (30), the mosquito-borne Japanese encephalitis virus strain SA-14-14-2 (32), and dengue virus strain PUO-280 (6) have a phenylalanine at this position. We therefore chose to make this conservative Leu-to-Phe change in addition to substituting a polar (Thr) and a charged (Asp) residue for Leu 107.Expression and secretion properties of mutants.
COS-1 cells
were transfected with recombinant plasmids encoding the modified E
proteins together with the prM protein, which is required for particle
assembly and maintenance of the conformation of E during transport and
secretion (3). All three of the mutants yielded RSPs with
a diameter of approximately 30 nm, which were secreted with essentially
the same efficiency as the wild-type control, indicating that the
various intermolecular interactions involving prM and E that are
necessary for particle assembly and secretion were not affected by the
mutations. Analysis of these particles showed that the prM proteins in
all cases were properly processed, and no differences in protein
composition, degree of maturation, particle density, or sedimentation
velocity were observed in comparison with wild-type RSPs (data not
shown). It was observed, however, that the Phe mutant (RSP-107F) had a
tendency to self-aggregate during the harvesting procedure, resulting
in somewhat reduced yields. Negatively stained electron micrographs of
purified wild-type and mutant RSPs are shown in Fig.
2.
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Fusion properties of mutant RSPs. Effects of the mutations on membrane fusion activity were assessed by mixing artificial liposomes with RSPs whose membranes had been fluorescently labeled in vivo with 1-pyrenehexadecanoic acid, acidifying the mixture, and continuously monitoring the decrease in pyrene excimer fluorescence caused by dilution of the probe in the target membrane (8).
Consistent with earlier results (8), RSP-wt, which contained the wild-type Leu at position 107, fused rapidly within the first seconds after acidification (Fig. 3). RSP-107F, with Phe at this position, also fused with liposomes at low pH, but both the initial rate and final extent of fusion were lower than with RSP-wt. The Thr mutant, RSP-107T, retained only a low level of fusion activity, and no fusion at all was observed with the Asp mutant, RSP-107D.
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Conformational changes at low pH.
To compare low-pH-induced
conformational changes in the E proteins of the wild-type RSP and the
nonfusing RSP-107D, we measured the binding activities of a panel of
E-specific MAbs whose epitopes in domains I, II, and III have been
mapped previously (36). As shown in the top panel of Fig.
4, these conformation-dependent epitopes
are altered by low-pH treatment, resulting in a characteristic change
in the MAb reactivity pattern of the wild-type E protein (42). With a few significant exceptions (see below), the
reactivity profiles with RSP-107D before and after low-pH treatment
(Fig. 4, middle) were very similar to those of the wild type,
indicating that the Leu-to-Asp mutation did not fundamentally impair
the switching mechanism or prevent the E protein from attaining its final low-pH conformation.
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Quaternary structure changes at low pH.
Earlier studies with
virions and RSPs have shown that the E protein is irreversibly
converted from a homodimer to a homotrimer upon exposure to low pH, and
these forms can be distinguished by sedimentation analysis after
solubilization with detergent (2, 42). To examine the
effect of the Leu 107-to-Asp mutation on this structural transition,
RSP-wt and RSP-107D were preincubated at pH 5.8 or 8.0, back-neutralized, solubilized with 0.5% Triton X-100, and analyzed by
sedimentation at pH 8.0 on sucrose gradients. As shown in Fig.
6, the pH 8.0 forms of the wild-type and
mutant E proteins exhibited identical sedimentation behavior,
confirming that the mutant protein is initially homodimeric. After
preexposure to low pH, a faster-sedimenting form of E, shown in earlier
studies to be a homotrimer (2, 42), was observed with both
RSP-wt and RSP-107D. In the case of RSP-107D, the majority of the E
protein had been converted to the trimeric form, but in three separate experiments the trimerization was less complete than it was with RSP-wt, suggesting that the 107D mutation, while still allowing trimers
to form, caused a decrease in trimerization efficiency. A similar
reduction was also observed with RSP-107T and, to a lesser extent, with
RSP-107F.
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Low-pH-induced association with lipid membranes.
To
investigate the initial low-pH-triggered interaction of the E protein
with the target membrane, we used a liposome coflotation assay in which
RSPs were mixed with liposomes at either pH 8.0 or 5.5 and then
analyzed by centrifugation in a sucrose step gradient (see Materials
and Methods). At pH 8.0, all of the E protein from both RSP-wt and
RSP-107D remained in the 20% sucrose layer, where it had been
initially applied (Fig. 7). After
treatment at pH 5.5, however, most of the wild-type E protein migrated
upward with the liposomes to the 5% sucrose layer, whereas essentially all of the protein carrying the 107D mutation remained in the 20%
sucrose layer (Fig. 7). This shows that RSP-107D does not form a stable
association with liposomes after low-pH treatment. In several
experiments it was found that the Phe and Thr mutants, unlike RSP-107D,
were able to interact with liposomes at low pH but to a lesser extent
than the wild type. Incomplete binding was always observed with
RSP-107F, whereas RSP-107T gave an irregular distribution of protein in
the sucrose gradient, suggesting that complexes of the 107T mutant with
liposomes were unstable and dissociated during centrifugation (data not
shown). This finding is consistent with the fusion data in Fig. 3 and
further suggests that substitutions at position 107 might directly
interfere with early interactions between the E protein and the target
membrane.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The data presented here provide functional evidence that a highly conserved loop at the tip of each subunit of the flavivirus E protein is important for fusion activity and may be directly involved in interactions with target membranes during the initial stages of membrane fusion. The notion that this portion of the protein serves as an internal fusion peptide is consistent with earlier observations that antipeptide antibodies recognizing the region from amino acids 98 to 110 are capable of blocking low-pH-induced fusion of TBE virus with artificial membranes (47) and react more strongly with the low-pH form of the dengue 2 virus E protein than with the native neutral form (40). We have recently observed that the flavivirus cross-reactive MAb A1, whose binding was shown in the present study to be abolished by amino acid substitutions at Leu 107, is also capable of inhibiting virus-liposome fusion and binding of isolated E proteins to artificial membranes (K. Stiasny et al., unpublished data). While all of these data suggest that the cd loop itself might insert into the target membrane during fusion, the actual contact region will ultimately have to be identified by more direct methods such as photolabeling with radioactive lipids (12, 18).
The tip of domain II is buried in the subunit interface of the
neutral-pH form of the E homodimer (Fig. 1) and is therefore inaccessible for membrane interactions in its native state. However, the quantitative conversion of E homodimers to homotrimers that occurs
at the pH of fusion would require at least a partial dissociation of
the native dimer, which could then lead to the exposure of the cd loop.
We have shown previously that truncated E dimers that lack the
stem-anchor region and therefore cannot trimerize nevertheless
dissociate at mildly acidic pH (46). It is likely that
ectodomain dissociation in the native virion is responsible not only
for initiating the reorganization of the viral envelope (dimer-trimer
transition) but also for a transient exposure of the fusion peptide in
a configuration that facilitates interaction with the target membrane.
Consistent with this idea, we have recently obtained evidence that
dimer dissociation is required for binding of E ectodomain fragments to
liposomes (Stiasny et al., unpublished). Subsequent steps in the fusion
reaction are likely to require more extensive conformational changes.
However, in contrast to the prevailing model based on the influenza
virus hemagglutinin and structurally related fusion proteins (24,
44, 48), it is difficult to envision the region containing the
TBE virus fusion peptide being converted to an extended 
-helical
structure at low pH, given the constraints placed upon it by the three
disulfide bridges in domain II, one of which (Cys 74-Cys 105) involves
the fusion peptide itself. Moreover, the extraordinarily rapid rate of
fusion by TBE virus (8) essentially rules out the
involvement of any steps preceding the lipid-mixing phase that are not
extremely favorable kinetically, such as the reduction of disulfide bonds.
The notion that an internal fusion peptide can reside in a disulfide-stabilized loop structure has already been supported by studies with avian sarcoma-leukosis virus. The fusion protein of this retrovirus contains a probable fusion peptide at an internal position starting about 21 amino acids from the N terminus (9, 23), and it was shown by mutagenesis that cysteine residues flanking this region are important for fusion activity (10).
Although the fusion proteins of influenza virus and flaviviruses have very dissimilar structures, it is intriguing that their fusion peptides both contain the tetrapeptide sequence GLFG. Mutagenesis studies with influenza virus hemagglutinin have demonstrated that at least the first glycine residue of this tetrapeptide is critically important for fusion (16, 35, 45). It is therefore likely that this motif has properties that are conducive to membrane interactions, a notion that is also supported by experiments with synthetic peptides (11, 33). A similar tetrapeptide sequence, GFLG, is found in the N-terminal fusion peptides of several retroviruses (11, 33) and hepatitis B virus (38). In the case of gp41 of human immunodeficiency virus, the conserved Phe appears to be important for maintaining the functionally active conformation of the fusion peptide (34). The sequence GFFG occurs naturally in a wild-type strain of the flavivirus Powassan virus at the position corresponding to GLFG in TBE virus (30), and we show here that TBE virus RSPs carrying this sequence (RSP-107F) are still capable of undergoing fusion with membranes, albeit less efficiently.
A number of viral fusion proteins appear to belong to a structural
superfamily whose members have in common the capability of adopting a
characteristic core structure involving a trimeric coiled coil of helices. These proteins all have either N-terminal or N-proximal fusion
peptides that reside at or near the tip of the coiled coil in the
fusion-active form. On the other hand, fusion proteins not belonging to
this structural class, i.e., those that are not cleaved and are not
predicted to form coiled coils, appear to have fusion peptides within
internal loop structures, distant from the N terminus, as has been
shown by mutagenesis (26, 28) in the case of the
alphaviruses and by both mutagenesis (14, 50) and direct
labeling experiments (12) in the case of rhabdoviruses.
The TBE virus E protein is currently the only viral fusion protein of
the non-coiled-coil type for which a high-resolution structure is
available and thus provides the first example of the native structure
of a probable internal fusion peptide. Comparison of the properties of
these functionally analogous but structurally and mechanistically
distinct protein classes should facilitate the identification of
essential features shared by both and thereby help to reveal some of
the fundamental principles common to protein-mediated fusion of membranes.
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ACKNOWLEDGMENTS |
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We thank Melby Wilfinger, Angela Dohnal, Walter Holzer, and Silvia Röhnke for excellent technical assistance.
Part of this work was supported by a grant from the International Human Frontier Science Program.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79505. Fax: 43-1-406-21-61. E-mail: steven.allison{at}univie.ac.at.
Present address: INTERCELL Biomedizinische Forschungs- und
Entwicklungs GmbH, A-1030 Vienna, Austria.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, May 2001, p. 4268-4275, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4268-4275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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75: 5627-5637
[Abstract] [Full Text]
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