From the Division of Biology, California Institute of Technology, Pasadena, California 91125
Received for publication, October 31, 2002
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ABSTRACT |
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The human immunodeficiency virus type 1 (HIV-1)
envelope glycoprotein is composed of a complex between the surface
subunit gp120, which binds to cellular receptors, and the transmembrane subunit gp41. Upon activation of the envelope glycoprotein by cellular
receptors, gp41 undergoes conformational changes that mediate fusion of
the viral and cellular membranes. Prior to formation of a fusogenic
"trimer-of-hairpins" structure, gp41 transiently adopts a
prefusogenic conformation whose structural features are poorly
understood. An important approach toward understanding structural
conformations of gp41 during HIV-1 entry has been to analyze the
structural targets of gp41 inhibitors. We have constructed epitope-tagged versions of 5-Helix, a designed protein that binds to
the C-peptide region of gp41 and inhibits HIV-1 membrane fusion. Using
these 5-Helix variants, we examined which conformation of gp41 is the
target of 5-Helix. We find that although 5-Helix binds poorly to native
gp41, it binds strongly to gp41 activated by interaction of the
envelope protein with either soluble CD4 or membrane-bound cellular
receptors. This preferential interaction with activated gp41 results in
the accumulation of 5-Helix on the surface of activated cells. These
results strongly suggest that the gp41 prefusogenic intermediate is the
target of 5-Helix and that this intermediate has a remarkably
"open" structure, with exposed C-peptide regions. These results
provide important structural information about this intermediate that
should facilitate the development of HIV-1 entry inhibitors and may
lead to new vaccine strategies.
Entry of the human immunodeficiency virus type 1 (HIV-1)1 into host cells is
mediated by its envelope glycoprotein, a complex of the
receptor-binding subunit gp120 and the transmembrane subunit gp41 (1).
gp41 possesses membrane fusion activity and contains an N-terminal
fusion peptide followed by two heptad repeat regions termed the
N-peptide region and the C-peptide region (see Fig. 1a) (1,
2). Upon binding of gp120 to its cellular receptors, CD4 and certain
chemokine co-receptors, gp41 undergoes a multi-step structural
transition from a native, nonfusogenic conformation to a fusion-active
conformation (3-7).
The presumed fusion-active conformation of gp41 is a complex of three
helical hairpins, in which each hairpin is formed from an N-peptide
helix and an anti-parallel C-peptide helix (see Fig. 1b)
(8-12). This "trimer-of-hairpins" structure is a six-helix bundle
consisting of an internal triple-stranded coiled-coil (three N-peptide
helices) with three outer C-peptide helices packed along hydrophobic
grooves. In this conformation, the fusion peptide and the transmembrane
segment are placed in closed proximity, providing a plausible mechanism
for apposition of the target and viral membranes (2, 9).
Synthetic C-peptides potently inhibit gp41-mediated membrane fusion
(13-15), and one of these, DP178, binds to a conformation of gp41
activated by interaction of envelope protein with cellular receptors
(5). These peptide inhibitors are unlikely to target the
trimer-of-hairpins structure, because of its extreme stability and the
high effective concentrations of the N- and C-peptide regions within a
hairpin. These observations suggest that native gp41 converts to a
transiently populated "prehairpin" intermediate prior to formation
of the fusogenic trimer-of-hairpins structure (see Fig. 6) (2). Other
entry inhibitors, such as IQN17 (16), N(CCG)-gp41 (17),
N36Mut(e,g) (18), and 5-Helix (19), may also bind this
intermediate, although there is no direct evidence for this hypothesis.
An understanding of the structural features of this prefusogenic
intermediate is critical because it appears to be an attractive drug
target (15, 20). Indeed, clinical trials have demonstrated the efficacy of the C-peptide DP178 (also called T-20) in reducing viral titers in
HIV-1-infected patients (21, 22).
5-Helix is a designed, recombinant protein (see Fig. 1, c
and d) that consists of three N-peptide regions and two
C-peptide regions (19). This protein is thought to bind the C-peptide region of gp41 and potently inhibits the fusogenic activity of gp41 at
nanomolar concentrations. However, it is unknown which conformation of
gp41 is the target of 5-Helix. In this study, we designed a tagged
5-Helix protein containing the Myc epitope (EQKLISEEDL) and
investigated the target of 5-Helix using a surface co-immunoprecipitation assay. Our results show that 5-Helix binds to
HIV-1 gp41 only after gp41-expressing cells come in contact with
cellular receptors, resulting in association of 5-Helix on the surface
of such cells. These results strongly suggest that 5-Helix binds to the
prehairpin intermediate of gp41 and that this intermediate is in an
"open" conformation that is accessible to inhibitory compounds.
Cloning and Mutagenesis--
The plasmid pMMHa3×Myc was used as
a template to amplify three tandem copies of the Myc epitope tag by PCR
with primers encoding both 5' and 3' NdeI sites (for
N-terminally tagged versions) and with primers encoding both 5' and 3'
BamHI sites (for C-terminally tagged versions). The
amplified 120-base pair fragments were ligated into the p5-Helix
plasmid to generate p3Myc/5-Helix and p5-Helix/3Myc, respectively.
Aspartate substitutions (V549D, L556D, Q563D, and V570D) in
p5-Helix/3Myc were introduced individually into the final (third) N40
segment by PCR to generate p5-Helix(D4)/3Myc. The p3Myc/6-Helix and
p6-Helix/3Myc constructs were prepared in the same manner as above by
ligating the PCR products into p6-Helix.
Protein Expression and Purification--
All of the proteins
were expressed in the Escherichia coli strain
BL21(DE3)/pLysS using the T7 expression system (23). Overnight cultures
(10 ml) were used to inoculate 1 liter of Luria Broth medium and grown
to log phase at 37 °C. Overproduction of the protein was induced by
the addition of isopropyl thio- CD Spectroscopy--
CD measurements were performed in
PBS (pH 7.4) with an Aviv 62DS CD spectrometer (Aviv Associates)
equipped with a thermoelectric temperature controller. Wavelength scans
(200-260 nm) were performed on 10 µM protein solutions.
Thermal denaturation profiles were obtained by measuring the
ellipticity at 222 nm ( Tissue Culture--
NIH3T3 and 293T cell lines were maintained
in Dulbecco's modified Eagle's medium supplemented with 1%
glutamine, 1% penicillin-streptomycin, and 10% fetal calf serum or
10% bovine calf serum, respectively, at 5% CO2 and
37 °C. Syncytia assays were performed as described previously
(15).
For the cell surface co-immunoprecipitation assays, 2 µg of the HIV-1
envelope expression plasmid pCMVgp160 was transfected by the calcium
phosphate method into 2 × 105 293T cells/well in a
six-well dish. Two days after transfection, the 293T cells were
incubated with 100 µg of each protein in the presence or absence of
receptors (10 µg of sCD4 or 3 × 106 target cells)
at 37 °C for 1 h. The cells were then washed with PBS (pH 7.4)
and lysed with 1.5 ml of lysis buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, and 1% Triton X-100). The clarified
supernatants were incubated overnight (4 °C) with 25 µl of
Sepharose beads coupled to the anti-Myc monoclonal antibody 9E10. After
four washes with PBS, the immunoprecipitates were separated by 10%
SDS-polyacrylamide gel electrophoresis and immunoblotted with the
anti-gp41 monoclonal antibody Chessie 8 (National Institutes of Health
AIDS Research and Reference Reagent Program), and a horseradish
peroxidase-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch).
To detect binding of 5- or 6-Helix to the cells, the immunoprecipitates
were separated on a 12% SDS-polyacrylamide gel and immunoblotted with
monoclonal antibody 9E10. The sCD4 and target cell lines (3T3.T4,
3T3.T4.CCR5, 3T3.T4.CXCR4) were obtained from the National Institutes
of Health AIDS Research and Reference Reagent Program.
C34 Peptide Binding Assay--
The C34 peptide precipitation
experiment was performed in 500 µl of PBS (pH 7.4) with 10 µM protein (5-Helix or 6-Helix variants) and 10 µM C34 peptide. The solution was added to 25 µl of
anti-Myc beads and incubated at 4 °C for 1 h. After the unbound
supernatant was removed, the beads were washed four times with 1.5 ml
of PBS buffer. The bead-bound samples were analyzed by a 16.5%
Tris-Tricine polyacrylamide gel (25) and immunoblotted with an anti-C34
polyclonal antibody and horseradish peroxidase-conjugated anti-rabbit
IgG antibody (Jackson ImmunoResearch).
Biophysical Characterization of 5- and 6-Helix Variants--
To
use 5-Helix as a structural probe for gp41, we designed two 5-Helix
variants containing three copies of the Myc epitope (EQKLISEEDL) at
either the N terminus (3Myc/5-Helix) or the C terminus (5-Helix/3Myc;
Fig. 1, c and d).
As a negative control, we constructed a Myc-tagged version of a mutant
5-Helix (5-Helix(D4)/3Myc) that contains a disruption of the
C-peptide-binding site and that was previously shown to be ineffective
in inhibiting gp41-mediated fusion (19). We also constructed N- and
C-terminally Myc-tagged versions of 6-Helix (3Myc/6-Helix and
6-Helix/3Myc, respectively), a protein that does not inhibit HIV-1
membrane fusion because the C-peptide-binding site has been filled by
an attached C-peptide.
Our two Myc-tagged versions of 5-Helix have biophysical properties
nearly identical to untagged 5-Helix (Table
I and Fig. 2). By CD analysis, all three proteins
show CD spectra with minima at 208 and 222 nm, as is typical for
helical proteins (Fig. 2a). The molar ellipticities at 222 nm for each of these proteins were indistinguishable, indicating
similar levels of helicity (Table I). Moreover, the three proteins were
highly soluble (data not shown) and extremely stable, with a melting
temperature (Tm) of 98 °C in the presence of
3.7 M guanidine hydrochloride in PBS (pH 7.4) (Table I and
Fig. 2b). We thus conclude that the Myc epitope tags do not
affect the overall helical structure and stability of 5-Helix protein.
Likewise, the biophysical properties of 6-Helix/3Myc and 3Myc/6-Helix
were nearly identical to untagged 6-Helix. Consistent with previous
results, the mutant construct 5-Helix(D4)/3Myc was slightly less
helical and less stable than wild-type 5-Helix (Table I) (19).
Inhibitory Activity of 5- and 6-Helix Variants--
Both
3Myc/5-Helix and 5-Helix/3Myc bound to synthetic C34 peptide in an
immunoprecipitation assay using the anti-Myc monoclonal antibody 9E10
(Fig. 3, lanes 3 and
4). In contrast, the 5-Helix(D4)/3Myc, 3Myc/6-Helix, and
6-Helix/3Myc proteins showed no C34 binding (lanes 5,
7, and 8).
Consistent with their C34 binding activity, both 3Myc/5-Helix and
5-Helix/3Myc showed potent inhibitory activity against cell-cell fusion
mediated by HIV-1 envelope protein, with half-maximal inhibition at 111 and 46 nM, respectively (Table I). These values are ~10- and 3-fold less potent than that of untagged 5-Helix. The reduced potency of the tagged proteins may be due to steric constraints in
accessing the gp41 C-peptide region during the membrane fusion process.
Furthermore, these constraints may be more severe for 3Myc/5-Helix, in
which the 39 additional residues of the epitope tag are expected to be
oriented toward the viral membrane (see Fig. 6). A reduction in
inhibitory activity has also been observed for a hemagglutinin
epitope-tagged version of the C-peptide DP178 (5). In contrast to the
inhibitory activity of the 5-Helix variants, four different control
proteins (5-Helix(D4)/3Myc, 6-Helix, 3Myc/6-Helix, and 6-Helix/3Myc)
showed no detectable inhibitory activity at the highest concentration
tested (10 µM).
Binding of Tagged 5-Helix to Activated gp41--
The preservation
of inhibitory activity in 3Myc/5-Helix and 5-Helix/3Myc allowed us to
explore the conditions under which 5-Helix binds to gp41. Using a
surface co-immunoprecipitation assay, we investigated whether these
proteins could bind to gp41 expressed on the surface of transfected
293T cells (Fig. 4a). 293T
cells were transfected with a HXB2 envelope expression plasmid, which
directs expression of gp160, the precursor polypeptide that is
proteolytically processed to generate the envelope complex of gp120 and
gp41 (26). 48 h after transfection, 293T cells were incubated with
the panel of 5-Helix and 6-Helix variants in the presence or absence of
sCD4. Binding of these polypeptides to the surface of transfected cells
was determined by immunoprecipitation with the anti-Myc monoclonal
antibody 9E10. Both 3Myc/5-Helix and 5-Helix/3Myc showed low levels of
gp41 binding in the absence of sCD4, and this binding was greatly
increased in the presence of sCD4 (lanes 4 and
6). The specificity of this gp41 binding was shown by the
lack of binding by 5-Helix(D4)/3Myc, 3Myc/6-Helix, and 6-Helix/3Myc
(lanes 8, 12, and 14). Interestingly,
a high molecular weight band, corresponding in size to uncleaved gp160, was also immunoprecipitated in a parallel pattern. Reprobing of the
Western blots with an anti-gp120 antibody confirmed that this band was
indeed gp160 (data not shown). Because the C-peptide DP178 also has
been shown to bind to receptor-activated gp160 (5), this result
indicates that significant conformational changes can be induced in the
envelope protein even in the absence of proteolytic processing. In
these immunoprecipitation experiments, we also detected an 80-kDa
background band whose identity is unknown. However, this band is
unrelated to HIV-1 envelope, because we also detect it in
immunoprecipitations from untransfected 293T cells (data not
shown).
We further investigated whether surface-expressed cellular receptors
could similarly activate the binding of 5-Helix to gp41. 293T cells
expressing gp160 were incubated with 5-Helix/3Myc in the presence of a
panel of retrovirally transduced NIH3T3 cell lines that express
cellular receptors for HIV-1 (Fig. 4b). Whereas nonexpressing NIH3T3 cells showed no effect (compare lanes 1 and 2), cell lines expressing CD4 triggered a large increase
in binding of 5-Helix/3Myc to gp41 (lanes 4-6). The
conformational changes allowing binding of 5-Helix/3Myc appear to be
largely induced by CD4, because the additional expression of CXCR4
(lane 6), the co-receptor for the envelope protein (HXB2
strain) used in these studies, caused only a slight increase in
binding. In all cases, the binding of gp41 by 5-Helix/3Myc was
effectively inhibited by the monoclonal antibody Q4120 (lanes
7-10), which binds to CD4 and blocks its binding by gp120. These
results demonstrate that 5-Helix binds to a conformation of gp41
activated by interaction of the envelope protein with cellular receptors.
Association of Tagged 5-Helix on the Surface of Transfected 293T
Cells--
To further characterize our surface immunoprecipitation
experiments, we determined the levels of 5-Helix/3Myc
immunoprecipitated from the cell surface in the presence and absence of
receptors. Low levels of 5-Helix/3Myc were precipitated in the absence
of sCD4 (Fig. 5a, lane 1), but the levels were
dramatically increased in the presence of sCD4 (lane 2). In
contrast, 5-Helix(D4)/3Myc and 6-Helix/3Myc showed no precipitation
either in the presence or absence of sCD4 (lanes 3-6).
The level of 5-Helix/3Myc precipitated was not affected by the addition
of NIH3T3 cells lacking human HIV-1 receptors (Fig. 5b,
lanes 1 and 2). In contrast, the levels of
precipitated 5-Helix/3myc were greatly enhanced by the addition of cell
lines expressing CD4 or CD4 and co-receptors (lanes 4-6).
In all cases, the immunoprecipitation of 5-Helix/3Myc was drastically
decreased by addition of the anti-CD4 antibody (Q4120) (lanes
7-10), paralleling the trend seen in gp41 binding experiments
(Fig. 4b). Together these results show that interaction of
envelope protein to CD4 facilitates association of 5-Helix to the cell surface.
Our results show that 5-Helix binds poorly to native gp41 but well
to gp41 activated by interaction of envelope protein with cellular
receptors. The fusogenic trimer-of-hairpins conformation is unlikely to
be disrupted by 5-Helix, because this hairpin structure involves
extremely stable intramolecular interactions (2). Consistent with this
assumption, we find that 5-Helix does not bind to recombinant 6-Helix,
which mimics the trimer-of-hairpins conformation of
gp41.2 Therefore, our results
strongly suggest that the molecular target of 5-Helix is the prehairpin
intermediate, a prefusogenic conformation previously inferred from the
inhibitory activity of C-peptides (2, 5).
Because this intermediate is a transiently populated species during the
membrane fusion process, it has been difficult to determine its
structure directly. The analysis of peptide inhibitors, however,
provides important insights into its structural features. The binding
of the C-peptide DP178 to this intermediate suggests that it contains
an exposed trimeric N-peptide coiled coil that has not yet formed a
six-helix structure with the adjacent C-peptide region of gp41 (5).
Interestingly, the N-peptide N36Mut(e,g), which forms a
homotrimer but cannot bind the C-peptide region of gp41, is also a
potent inhibitor of gp41-mediated fusion (18). It remains to be shown
directly that N36Mut(e,g) binds to the prehairpin
intermediate, but this observation raises the possibility that the
trimeric N-peptide region of the gp41 prehairpin intermediate undergoes
a monomer-trimer equilibrium (18,
27).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactoside to a final concentration of 1 mM. After 3 h of
induction, the cells were harvested and stored frozen (
20 °C)
until purification. Bacterial pellets were resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM
NaCl, lysed by sonication, and centrifuged (6,000 × g
for 30 min) to obtain an inclusion body fraction. Each 5-Helix and
6-Helix variant was enriched in this fraction. The pellets were
resuspended in 100 mM Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride, 10 mM imidazole, and
1 mM dithiothreitol. After clarification by centrifugation
(15,000 × g for 15 min), the histidine-tagged proteins
were affinity-purified on nickel-nitrilotriacetate (Qiagen) columns at
room temperature. The proteins were eluted with 50 mM
Tris-HCl buffer (pH 8.0) containing 6 M urea, 100 mM NaCl, and 100 mM imidazole. The eluted
protein was refolded by dialyzing against 50 mM Tris-HCl
(pH 8.0). After dialysis, all of the proteins were purified to >95%
purity by anion exchange chromatography on an ÄKTA Purifier 10 (Amersham Biosciences), using a MonoQ column and a linear gradient of
NaCl (0-1 M). Protein concentrations were determined by
absorbance at 280 nm in 6 M guanidine hydrochloride
(24).
222) as a function of
temperature. Protein solutions (10 µM) were measured at
1-degree intervals starting at 4 °C, with an equilibration time of 2 min and an acquisition time of 0.5 min. The apparent melting
temperature (Tm) was estimated from the maximum
of the first derivative of
222 with respect to
temperature. Because of the extreme thermal stability of these
proteins, these experiments were performed in PBS in the presence of
3.7 M guanidine hydrochloride.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
HIV-1 gp41 structure and 5-Helix variant.
a, a schematic view of HIV-1 gp41, showing the location of
the fusion peptide (FP), the two hydrophobic heptad repeat
regions, the transmembrane segment (TM), and the cytoplasmic
region (cyto). The location of certain N- and C-peptides are
drawn above. b, a ribbon model of the N36/C34 complex (8),
which forms the core of the gp41 ectodomain, viewed looking from the
side (left panel) and down the 3-fold axis (right
panel). Molecular graphics were produced using WebLab Viewer
(Molecular Simulations). c, a 5-Helix variant (5-Helix/3Myc)
that consists of three N40 segments (gray), two C38 segments
(blue), a His6 tag, and a 3Myc epitope tag
(green). The N40 to C38 junctions are joined with a GGSGG
linker, and the C38 to N40 junctions are joined with a GSSGG linker.
d, a structural model of 5-Helix/3Myc based on the known
structure of the N36/C34 complex (8). Note that 5-Helix and its
variants lack the third C-peptide helix and therefore bind to free
C-peptide with high affinity.
Biophysical data and inhibitory potency of 5- and 6-helix variants
222). The apparent melting
temperatures (Tm) were estimated from thermal
denaturation profiles of
222. Inhibition of cell-cell fusion
was measured in a syncytium assay. The means and standard errors were
calculated from triplicate experiments.
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Fig. 2.
Helical structure and thermal stability of
5-Helix variants. a, CD spectra of 5-Helix ( ),
3Myc/5-Helix (
), and 5-Helix/3Myc (
) at 4 °C in PBS (pH 7.4).
b, thermal denaturation profiles of 5-Helix (
),
3Myc/5-Helix (
), and 5-Helix/3Myc (
) monitored by ellipticity at
222 nm in PBS (pH 7.4) containing 3.7 M guanidine
hydrochloride.
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Fig. 3.
C34 peptide binding assay of the 5- and
6-Helix variants. C34 peptide was mixed with the 5- and 6-Helix
variants and immunoprecipitated with an anti-Myc monoclonal antibody.
Binding to C34 peptide was determined by Western blot analysis with an
anti-C34 polyclonal antibody.
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Fig. 4.
Cell surface co-immunoprecipitation of 5- and
6-Helix variants. a, 293T cells transfected with gp160 were
incubated with 5-Helix (lanes 1-8) and 6-Helix (lanes
9-14) variants in the absence or presence of sCD4 and subjected
to cell surface co-immunoprecipitation with the anti-Myc monoclonal
antibody 9E10. Binding to gp41 was determined by Western blot analysis
with the gp41 monoclonal antibody Chessie 8. b, 293T cells
expressing gp160 were incubated with 5-Helix/3Myc in the absence or in
the presence of retrovirally transduced NIH3T3 cell lines that express
cellular receptors for HIV-1. Binding to gp41 was determined as in
a. In lanes 7-10, the anti-CD4 monoclonal
antibody Q4120 was added to the reactions to block CD4 interactions
with gp120. The arrows indicate the positions of gp41 and
gp160. The background band (*) at ~80 kDa is unrelated to gp120/gp41,
as explained in the text.
DISCUSSION
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REFERENCES
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Fig. 5.
Cell surface localization of 5- and 6-Helix
variants. a, 293T cells transfected with gp160 were
incubated with 5- (lanes 1-4) and 6-Helix (lanes
5 and 6) variants in the absence or presence of sCD4
and subjected to cell surface immunoprecipitation with the anti-Myc
monoclonal antibody 9E10. Association of the protein to the transfected
cell surface was determined by Western blot analysis with the anti-Myc
monoclonal antibody 9E10. b, 293T cells expressing gp160
were incubated with 5-Helix/3Myc in the absence or in the presence of
retrovirally transduced NIH3T3 cell lines that express cellular
receptors for HIV-1. Association of the protein to the cell surface was
determined as in a. In lanes 7-10, the anti-CD4
monoclonal antibody Q4120 was added to block CD4 interactions with
gp120. The positions of protein molecular mass markers are
indicated at the left.
Our findings indicate that this prefusogenic conformation also contains
C-peptide regions that are exposed enough to allow access of tagged
5-Helix, a 30-kDa protein. Although this exposed C-peptide region
almost certainly binds to the 5-Helix protein as an -helix, we
cannot exclude the possibility that it exists in a different
conformation in the prehairpin intermediate and is induced into a
helical conformation by binding of 5-Helix. Taken together, these
results provide a view of the prehairpin intermediate as a largely open
structure in which both the N- and C-peptide regions are accessible and
thus vulnerable to binding by peptide inhibitors (Fig.
6).
|
Treatment of HIV-1 virions with sCD4 has been shown to result in gp120 dissociation and the increased binding of several gp41 monoclonal antibodies (4). This observation raises the formal possibility that increased binding of 5-Helix to activated gp41 may likewise reflect exposure of large regions of gp41 because of gp120 shedding. However, we believe that the target of 5-Helix is likely to be a true fusion intermediate, because binding leads to inactivation of gp41 fusion activity. Furthermore, we find that sCD4 leads to increased binding of 5-Helix to uncleaved gp160 (Fig. 4), a result incompatible with the view that gp120 shedding is responsible for 5-Helix binding.
We find that low levels of gp41 binding are observed in the absence of cellular receptors (Fig. 4, a, lanes 3 and 5, and b, lanes 1 and 2), implying that the C-peptide region of gp41 is only partially accessible in the absence of activation. This poor accessibility in native gp41 may be due to steric constraints or the adoption of an alternative conformation by the C-peptide region. Alternatively, the low level detected may reflect metastability of the native state of viral envelope proteins (28-30); that is, a small fraction of gp41 molecules at a given time may adopt a prehairpin-like conformation even in the absence of cellular receptors. It is interesting to compare our results with the finding that several monoclonal antibodies (2F5, 4E10, and Z13) directed against the membrane proximal region of gp41 bind better to native gp41 than activated gp41 (31). The epitopes recognized by these antibodies are located slightly C-terminal to the end of the C-peptide, because the 2F5 epitope spans residues 657-670 and the 4E10 epitope spans 671-676. It is likely that these differences in binding preference reflect different conformations recognized by 5-Helix versus the monoclonal antibodies. The study of these differences may reveal structural changes in gp41 during the fusion process.
These insights into HIV-1 gp41-mediated membrane fusion will likely also apply to the entry of many other enveloped viruses that have fusion proteins with heptad repeats. Structural studies have revealed that members of the retrovirus, orthomyxovirus, paramyxovirus, and filovirus families contain fusion proteins with a trimer-of-hairpins structure (1, 32). Beyond these structural similarities, the paramyxovirus SV5 fusion protein (F) has been shown to form a transient prehairpin intermediate prior to fusion that can be inhibited by synthetic peptides corresponding to the heptad repeats (33).
Finally, it should be noted that the N- and C-peptide regions of the
gp41 prefusogenic intermediate are highly conserved among diverse HIV-1
strains (8, 19). Therefore, it is not surprising that agents targeting
these regions have broad neutralizing activity, although the emergence
of inhibitor-resistant strains remains a concern (34). Our results
should facilitate the development of HIV-1 entry inhibitors and may
lead to new vaccine strategies.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. Michael J. Root and Peter S. Kim for providing the p5-Helix and p6-Helix vectors. We also thank Dr. Peter S. Kim for critical reading of the manuscript and members of the Chan lab for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 7 PO1 GM56552-05 and by a Burroughs Wellcome Fund Career Development Award in Biomedical Sciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Postdoctoral fellow of the Japan Society for the Promotion of Science.
§ Bren Scholar. Rita Allen Scholar. To whom correspondence should be addressed: Div. of Biology MC114-96, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125. Tel.: 626-395-2670; Fax: 626-395-8826; E-mail: dchan@caltech.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M211154200
2 T. Koshiba and D. C. Chan, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; PBS, phosphate-buffered saline; sCD4, soluble CD4; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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