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Journal of Virology, October 1999, p. 8578-8586, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic
Cavity in Membrane Fusion
Hong
Ji,1
Wei
Shu,1
F. Temple
Burling,1
Shibo
Jiang,2 and
Min
Lu1,*
Department of Biochemistry, Weill Medical
College of Cornell University,1 and
Lindsley F. Kimball Research Institute, New York Blood
Center,2 New York, New York 10021
Received 19 January 1999/Accepted 14 June 1999
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ABSTRACT |
The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an 
-helical core structure responsible for
mediating membrane fusion during viral entry. Recent studies suggest
that a conserved hydrophobic cavity in the coiled coil of this core
plays a distinctive structural role in maintaining the fusogenic
conformation of the gp41 molecule. Here we investigated the importance
of this cavity in determining the structure and biological activity of
the gp41 core by using the N34(L6)C28 model. The high-resolution
crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective
mutants reveal that each mutant sequence is accommodated in the
six-helix bundle structure by forming the cavity with different sets of
atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective
inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than
the wild-type molecule. The enhanced inhibitory activity by
fusion-defective mutations correlates with local structural
perturbations close to the cavity that destabilize the six-helix
bundle. Taken together, these results indicate that the conserved
hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1
entry and its inhibition and provides a potential antiviral drug target.
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INTRODUCTION |
Infection by retroviruses and other
enveloped viruses is initiated by fusion of the viral and cellular
membranes, with the subsequent delivery of the viral genome into the
host cell. Viral envelope glycoproteins play a critical role in this
infectious process, because membrane fusion is an essential step for
viral entry. They are also major targets in attempts to elicit the
antiviral immune response in infected hosts. The envelope glycoprotein
of human immunodeficiency virus type 1 (HIV-1) consists of two
noncovalently associated subunits, gp120 and gp41, which are displayed
on the lipid bilayer of the virion as well as on the plasma membranes of infected cells (42). The envelope glycoprotein is
responsible for promoting membrane fusion between the virus and target
cell during infection and between infected cells and uninfected cells during multinucleated giant cell (syncytium) formation (44). Binding of the surface gp120 subunit to the CD4 glycoprotein and a
chemokine receptor on the T-cell surface triggers the membrane fusion
activity of the transmembrane gp41 subunit (17, 37, 43, 44,
50). This receptor binding-mediated activation of membrane fusion
is postulated to involve a conformational change in the envelope
protein complex from a native (nonfusogenic) to a fusion-active
(fusogenic) state. This conformational transition likely facilitates
exposure of the hydrophobic, glycine-rich sequence referred to as the
fusion peptide at the amino terminus of gp41, leading to insertion into
the bilayer of the target membrane and initiating fusion (2, 6,
26).
The mechanism by which the gp41 molecule mediates membrane fusion has
been the subject of intense investigation (8). As seen in
most viral fusion proteins, the fusion peptide region of the gp41
ectodomain is followed by two regions with a 4-3 hydrophobic (heptad)
repeat, a sequence feature characteristic of coiled coils (Fig.
1) (7, 16, 24). Protein
dissection studies demonstrated that these two heptad-repeat regions
from a soluble, -helical core consisting of a trimer of antiparallel
dimers (39, 41). Crystallographic analysis of this gp41 core
confirmed that it is a six-helix bundle (10, 56, 57). The N
(amino)-terminal helices form an interior trimeric coiled coil, while
the C (carboxyl)-terminal helices pack in an antiparallel manner into
three highly conserved hydrophobic grooves on the surface of the coiled
coil. Given that this core structure is exceedingly stable to thermal
denaturation and resembles the fusion-pH-induced conformation of
influenza virus hemagglutinin, it was suggested that the six-helix
bundle represents the conformation of fusion-active gp41 (10, 41, 56, 57). This view has been confirmed by recent antibody-binding studies which show that a monoclonal antibody specifically recognizing the gp41 core binds to the surface of HIV-1-infected cells only after
interaction of the envelope protein complex with soluble CD4
(29).
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FIG. 1.
Schematic diagram of HIV-1 gp41. The important
functional features of the gp41 ectodomain and the amino acid sequences
of the N34 and C28 segments are shown. The N34(L6)C28 model consists of
N34 and C28 plus a six-residue linker. Two point mutations, L568A and
W571R, that abolish membrane fusion are indicated above the sequence.
The disulfide bond and four potential N-glycosylation sites are
depicted. The residues are numbered according to their position in
gp160.
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Synthetic peptides corresponding to the N- and C-terminal helical
regions of the gp41 ectodomain have been shown to be potent inhibitors
of HIV-1 infection and syncytium formation (30, 60, 62). It
is striking that the C peptide T-20 has high efficacy in suppressing
HIV-1 replication in clinical trials (35). Although the
mechanism of action of these peptide inhibitors is not known, considerable evidence suggests that they act by binding to viral gp41,
through a dominant-negative mechanism, to block formation of its
fusion-active core (10, 11, 23, 34, 41, 45, 61). The
structural and biophysical characteristics of the gp41 core and
experiments on the inhibition of HIV-1 infectivity by gp41 peptides
have led to the proposal that formation of the helical structure brings
the viral and cellular membranes into close proximity and thus
overcomes the energy barrier for membrane fusion (23, 27,
57).
The trimeric coiled-coil surface of the gp41 core is highly grooved and
possesses a conserved hydrophobic cavity that is important for packing
interactions between the N- and C-terminal helices (10).
Mutations in the Leu 568 and Trp 571 residues, which are involved in
the formation of this cavity, abolish membrane fusion activity
(5). Here we have tested the importance of this cavity in
determining the structure and function of the gp41 core by assessing
the effects of Leu 568-to-Ala and Trp 571-to-Arg substitutions in the
N34(L6)C28 model. The X-ray crystal structures of two single mutants at
resolutions of 1.6 Å (L568A) and 2.1 Å (W571R) show that each mutant
sequence is accommodated in the six-helix bundle structure by forming
the cavity with different sets of atoms. Remarkably, L568A and W571R
are effective inhibitors of HIV-1 infection, and they exhibit 16- and
5-fold greater activities than N34(L6)C28, respectively. Moreover, the
double mutant (L568A/W571R) has the highest activity, 35-fold greater
than that of the wild-type molecule. Our results provide evidence for
the hypothesis that the conserved hydrophobic cavity in the coiled coil
of the gp41 core plays a key role in HIV-1 entry and its inhibition.
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MATERIALS AND METHODS |
Protein production and purification.
Mutations were
introduced into the N34(L6)C28 model by single-stranded mutagenesis
(36) and verified by DNA sequencing. Standard recombinant
DNA techniques were used (51). All recombinant peptides were
expressed in Escherichia coli BL21(DE3)/pLysS by using the
T7 expression system (54). Cells, freshly transformed with
an appropriate plasmid, were grown to late log phase. Protein expression was induced by addition of 0.5 mM
isopropylthio-
-D-galactoside (IPTG). After another
3 h of growth at 37°C, the bacteria were harvested by
centrifugation, and the cells were lysed with glacial acetic acid as
described previously (40). Peptides were purified from the
soluble fraction to homogeneity by reverse-phase high-performance liquid chromatography, using a Vydac C18 preparative column
and a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. Peptide identity was confirmed by mass spectrometry. Peptide concentrations were determined by absorbance at 280 nm in the presence
of 6 M guanidinium hydrochloride (19).
CD spectroscopy.
Circular dichroism (CD) spectra were
acquired at a peptide concentration of 10 µM in 50 mM sodium
phosphate (pH 7.0)-150 mM NaCl (phosphate-buffered saline [PBS])
with an Aviv 62 DS spectrometer as described previously
(40). The wavelength dependence of molar ellipticity,
[
], was monitored at 0°C as the average of five scans, using a
5-s integration time at 1.0-nm wavelength increments. Spectra were
baseline corrected against the cuvette with buffer alone. The helix
content was estimated from the CD signal by dividing the mean residue
ellipticity at 222 nm by the value expected for 100% helix formation
by helices of comparable size, i.e., 
33,000° cm2
dmol
1 (13). Thermal stability was determined
by monitoring the change in the CD signal at 222 nm as a function of
temperature, and thermal melts were performed at intervals of 2°C
with a 2-min equilibration at the desired temperature and an
integration time of 30 s. Reversibility was checked by repeated
scans. The thermal melts of L568A and W571R were reversible, while
those of N34(L6)C28 and L568A/W571R were not reversible. The midpoint
of the thermal unfolding transition (apparent melting temperature
[Tm]) was determined from the maximum of the
first derivative, with respect to the reciprocal of the temperature, of
the []222 values (4). The error in
estimation of Tm is ±1°C.
Equilibrium ultracentrifugation.
Sedimentation equilibrium
analysis was performed on a Beckman XL-A analytical ultracentrifuge as
described previously (40). Protein solutions were dialyzed
overnight against PBS; loaded at initial concentrations of 10, 30, and
100 µM; and analyzed at rotor speeds of 20 and 23 krpm at 20°C.
Data sets were fitted simultaneously to a single-species model with the
program NONLIN (31). The protein partial specific volume and
solvent density were calculated with constants from Laue et al.
(38). Molecular weights were all within 10% of those
calculated for an ideal trimer, with no systematic deviation of the residuals.
Crystallization, data collection, and structure
determination.
The N34(L6)C28 variants were crystallized by
hanging-drop vapor diffusion at room temperature. To grow crystals, a
10-mg/ml stock of peptides was diluted 1:1 with a reservoir and allowed to equilibrate against the reservoir solution. Initial crystallization conditions were screened by using sparse matrix crystallization kits
(Crystal Screen I and II; Hampton Research, Riverside, Calif.) and then
optimized. Crystals of L568A in space group R3 (a = b = 49.68 Å, c = 60.20 Å) were grown from
0.1 M sodium acetate (pH 4.6)-0.2 M ammonium sulfate-20% (vol/vol)
glycerol and transferred to a cryoprotectant solution containing 25%
(vol/vol) glycerol in the corresponding mother liquor. Crystals of
W571R in space group R3 (a = b = 53.39 Å;
c = 59.56 Å) were obtained from 0.1 M sodium acetate
(pH 4.6)-0.2 M ammonium sulfate-17% polyethylene glycol 4000-20%
(vol/vol) glycerol. Cryoprotected crystals were frozen in propane
before data collection. Data were collected at 95 K by using an R-axis
IV image plate detector mounted on a Rigaku RU200 rotating anode X-ray
generator at the X-ray Crystallography Facility at the Weill Medical
College of Cornell University. All diffraction data were integrated and
scaled with the HKL suite (48).
The structures of L568A and W571R were determined by molecular
replacement with AMoRe (46). The 2.4-Å structure of the
wild-type N34(L6)C28 trimer (PDB code ISTZ) was used in a combined
rotation-translation search (with data for 15.0 to 3.5 Å) to yield
solutions for L568A (correlation coefficient = 66.8%;
R factor = 47.9%) and for W571R (correlation
coefficient = 74.8%; R factor = 38.9%). The
models were refined with simulated annealing and atomic displacement parameter refinements by using the program X-PLOR (1). The models were rebuilt to reflect the sequences of L568A and W571R by
using conventional (2Fo Fc)
calc and
(Fo Fc)calc maps with the program O
(33). Atomic coordinates for L568A (Protein Data Bank
[PDB] file name 1QR9) and W571R (PDB file name 1QR8) have been
deposited in the PDB.
Cell-cell fusion and infectivity assays.
The inhibitory
activities of N34(L6)C28 and variants thereof were determined by using
a dye transfer fusion assay as described previously (28).
HIV-1IIIB-infected H9 cells were labeled with 2',7'-bis-(2-carboxyethyl)-5 (and 6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM). Fluorescently labeled HIV-1IIIB-infected
H9 cells (104) and MT-2 cells were cocultured at a 1:10
ratio at various peptide concentrations for 2 h at 37°C in a
96-well microplate to obtain a dose-response curve. The fused cells
were scored for dye transfer under fluorescence microscopy. In the
infectivity assay, as described previously (28),
HIV-1IIIB was inoculated with 104 MT-2 cells at
multiplicity of infection of 0.0045 in RPMI 1640 containing 10% fetal
bovine serum in the presence of various peptide concentrations. After
incubation at 37°C for 1 and 24 h, half of the culture medium
was changed. HIV-1IIIB-mediated cytopathic effect (CPE) was
assessed by viability assay 6 days after infection.
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RESULTS |
Characterization of the mutant N34(L6)C28 cores.
The
recombinant N34(L6)C28 model of the gp41 ectodomain core, consisting of
residues 546 to 579 and 628 to 655 joined by a linker of six
hydrophilic residues (Fig. 1), forms a stable six-helix bundle
(40, 56). Earlier genetic studies indicate that the Leu
568-to-Ala and Trp 571-to-Arg substitutions inhibit membrane fusion
(5). X-ray crystallographic analyses of the gp41 core show
that these two residues are involved in forming a large hydrophobic cavity on the surface of the trimeric coiled coil (10, 56, 57). To define the structural and functional importance of these residues and, hence, the cavity in promoting membrane fusion, we
replaced Leu 568 with Ala and Trp 571 with Arg in N34(L6)C28, either
separately or simultaneously, to yield two single mutants (L568A and
W571R) and a double mutant (L568A/W571R).
The CD spectra of L568A, W571R, and L568A/W571R exhibit the
characteristic signature of an
-helical conformation, with minima
at
222 and 208 nm (Fig.
2A). Each folded
mutant core contains
>90%
-helical structure at 0°C in PBS at a
peptide concentration
of 10 µM. Under these conditions, the apparent
Tms of N34(L6)C28
(wild type), L568A, W571R, and
L568A/W571R are 70, 56, 61, and
54°C, respectively (Fig.
2B; Table
1). Thus, the mutant molecules
are less
stable than the wild-type counterpart. Moreover, sedimentation
equilibrium demonstrates that all the three mutant cores are trimeric
in solution (Fig.
2C; Table
1). These results indicate that the
Leu
568-to-Ala and Trp 571-to-Arg mutations do not alter structural
features sufficiently to disrupt the six-helix bundle formation.
The
destabilization effect of these mutations observed here may
be related
to the fusion-defective phenotype (see below).
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FIG. 2.
Folding of the N34(L6)C28 mutants as helical trimers.
(A) CD spectra of L568A (squares), W571R (triangles), and L568A/W571R
(circles) at 0°C in PBS (pH 7.0) at a peptide concentration of 10 µM. (B) Thermal melts monitored by CD at 222 nm for L568A (squares),
W571R (triangles), and L568A/W571R (circles) in PBS (pH 7.0) at a
peptide concentration of 10 µM. (C) Sedimentation equilibrium studies
of the mutant N34(L6)C28 cores indicate that all species are trimeric.
Representative analytical ultracentrifugation data (20 krpm) for
L568A/W571R collected at 20°C in PBS (pH 7.0) at a peptide
concentration of ~30 µM are shown. The natural logarithm of the
absorbance at 280 nm is plotted against the square of the radial
position. Deviations from the calculated values are plotted as
residuals in the upper panel.
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Crystal structures of L568A and W571R.
To obtain a
high-resolution view of the conserved hydrophobic cavity in the mutant
cores, the X-ray crystal structures of L568A and W571R were determined.
Crystals of the two mutants were grown by hanging-drop vapor diffusion
(see Materials and Methods). The L568A and W571R structures were
determined by molecular replacement, using a 2.4-Å structure of
N34(L6)C28 (56). The electron density map of L568A allows
the completion of the structure of 68 amino acid residues and the
placement of 49 water molecules (Fig. 3A and B), while that of W571R reveals the positions of all of the amino
acid residues except a few disordered side chains at the helix termini
and in the linker region (Fig. 3C and D). The structure of L568A was
refined against 15.0- to 1.6-Å data to a 1.6-Å resolution to yield
crystallographic and free R factors of 20.2 and 26.1%, respectively (Table 2). The structure of
W571R was refined to a 2.1-Å resolution, with crystallographic and
free R factors of 21.2 and 26.2%, respectively, in the
resolution range of 15.0 to 2.1 Å (Table 2). Details of the data
collection and refinement statistics are presented in Table 2.
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FIG. 3.
Electron density maps at the substitution sites. (A)
Initial 2Fo Fc
map of L568A after density modification and phase improvement with DM
(14). The initial molecular replacement solution model is
superimposed. (B) Final 2Fo Fc map of L568A with the refined model
superimposed. (C) Initial 2Fo Fc map of W571R as described for panel A. (D)
Final 2Fo Fc map
of W571R as described for panel B. The side chains of the mutated
residues are indicated by white arrows. Water molecules are indicated
by small red balls. The maps of L568A and W571R are contoured at 1.8 and 1.2  , respectively. Figures were generated with the program O
(33).
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The overall structures of L568A and W571R are very similar to that of
the wild-type core. In all cases, three hairpin-like
molecules pack
together on the crystallographic threefold-symmetry
axis to form a
six-helix bundle. The N-terminal helices within
the bundle form a
parallel, three-stranded coiled coil in the
characteristic acute
"knobs-into-holes" arrangement (Fig.
4) (
15,
25). Three C-terminal
helices pack in an antiparallel orientation
into three hydrophobic
grooves on the surface of the central coiled-coil
trimer (Fig.
4). The
Leu 568 and Trp 571 residues replaced in
L568A and W571R are at each of
the grooves, where they form the
right wall of a deep cavity that
accommodates the side chains
of two tryptophans and a isoleucine from
the C-terminal helices
(Fig.
5). The root
mean square (rms) deviations between all C
atoms of the
trimeric coiled coil in the wild-type and mutant
molecules are 0.43 Å for L568A and 0.27 Å for W571R. The C-terminal
helices in L568A and
W571R can also be superimposed upon the wild-type
counterpart with rms
deviations of 0.78 and 0.52 Å, respectively.
Thus, it appears that the
fusion-defective Leu 568-to-Ala and
Trp 571-to-Arg mutations each do
not affect the overall protein
fold of the gp41 core.
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FIG. 4.
Overall views of the mutant L568A and W571R cores. (A)
Side view. The amino termini of N34 and the carboxyl termini of C28 are
at the top. Helices in L568A (yellow) and W571R (pink) were used for
the superposition. The bottom of the central N34 coiled-coil surface
contains three symmetry-related hydrophobic cavities (one is outlined
by the box). (B) View from the top, looking down the threefold axis of
the trimer. The same color coding as in panel A is used. Figures were
generated with the program SETOR (21).
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FIG. 5.
The conserved hydrophobic cavity in the L568A and W571R
structures. (A) Cross-section of helix packing near the conserved
cavity in L568A. The structures of the wild-type molecule (red) and
L568A (green) are overlaid. The side chains of the mutated residues are
in yellow. (B) Cross-section of helix packing in W571R. The same
superposition as in panel A was used. (C) Interactions of the C28 helix
with a deep cavity on the surface of the N34 coiled coil in L568A. The
C28 helices of the wild-type molecule (red) and L568A (green),
represented as ribbons, are shown against a surface representation of
the N34 coiled coil in L568A. (D) Interactions of the C28 helix with a
deep cavity on the surface of the N34 coiled coil in W571R. The same
superposition as in panel C was used. Figures were generated with the
programs SETOR (21) and GRASP (47).
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The replacement of Leu 568 with Ala leads to local structural
rearrangements in packing interactions between the N34 and C28
helices;
the side chains of several key residues, including Tyr
638, Glu 634, Ile 635, Trp 631, and Trp 628, deviate substantially
near the conserved
cavity (Fig.
5A and C). In contrast, the Arg
571 residue in W571R is
accommodated without significantly altering
the six-helix bundle
structure. A local distortion occurs at Trp
628 (Fig.
5B and D), which
has high atomic
B factors and may be
poorly ordered due to
chain terminus effects. In addition, the
Arg 571 side chain is oriented
toward the carboxyl group of the
Glu 632 side chain from the abutting
C28 helix, and the two side
chains can interact via a water-mediated
hydrogen bond (2.81 and
2.70 Å). This polar interaction appears to be
responsible for
the ordering of the Arg 573 side
chain.
Effect of fusion-defective mutations on HIV-1 inhibition.
We
have previously demonstrated that N34(L6)C28 can block syncytium
formation at micromolar concentrations (40). To test if the
Leu 568-to-Ala and Trp 571-to-Arg mutations affect this inhibitory
activity, we examined the relative abilities of L568A, W571R, and
L568A/W571R to inhibit HIV-1 infection by using both cell-cell fusion
and infectivity assays. Figure 6 shows
the inhibition of HIV-1IIIB-infected H9 cell-mediated
syncytium formation and HIV-1 infectivity by different concentrations
of the wild-type and mutant N34(L6)C28 molecules. The double mutant is
strikingly the most effective inhibitor, with 50% inhibitory
concentrations (IC50s) of 48 nM for syncytium formation and
44 nM for CPE, compared with IC50s of 1.51 and 1.49 µM,
respectively, for N34(L6)C28 (Fig. 6; Table
3). L568A also potently inhibits
cell-cell fusion and infection, with IC50s of 95 and 82 nM,
respectively, while W571R does so with IC50s of 0.25 and
0.32 µM, respectively (Fig. 6; Table 3). Thus, the fusion-defective
mutations cause the N34(L6)C28 core to have 5-, 16-, and 35-fold
greater inhibitory activity for W571R, L658A, and L568/W571R,
respectively.
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FIG. 6.
Inhibition of HIV-1 infection by N34(L6)C28 variants.
(A) Inhibition of HIV-1IIIB-infected H9 cell-induced
cell-cell fusion by N34(L6)C28 (diamonds), L568A (triangles), W571R
(squares), and L568A/W571R (circles). Error bars indicate standard
deviations from quadruplicate experiments. (B) Inhibition of
HIV-1IIIB-mediated CPE by N34(L6)C28 (diamonds), L568A
(triangles), W571R (squares), and L568A/W571R (circles). Error bars
indicate standard deviations from triplicate experiments.
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In contrast to the Leu 568-to-Ala and Trp 571-to-Arg mutations, the Ser
substitution for Ile 573, an
a heptad position,
essentially
disrupts the six-helix complex formation (
40). Consequently,
N34(L6)C28 bearing this mutation inhibits cell-cell fusion and
infection with IC
50 values of 0.69 and 0.73 µM,
respectively (Table
3). This activity appears to reflect that of the
unfolded C28
segment in the I573S mutant, since the isolated N34 and
C28 peptides
have IC
50s of 9.74 and 0.95 µM for syncytium
formation and 11.38
and 2.19 for CPE, respectively (Table
3). To
examine whether
the Ile 573-to-Ser mutation can eliminate the antiviral
activity
of L568A/W571R, we replaced Ile 573 with Ser in the double
mutant
to produce a triple mutant (L568A/W571R/I573S). CD experiments
indicate that the triple mutant is predominantly unfolded at 0°C
in
PBS at a peptide concentration of 10 µM (Fig.
7A; Table
1).
Interestingly, this
molecule is significantly less active in blocking
cell-cell fusion and
infectivity, with IC
50s of 0.77 and 1.15
µM, respectively
(Fig.
7B; Table
3). Taken together, these results
indicate that the
inhibition of HIV-1 infection by the N34(L6)C28
core is both
conformation and sequence specific and that the L568
and W571 residues
are important determinants of the inhibitory
activity.
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FIG. 7.
An Ile 573-to-Ser mutation disrupts the six-helix bundle
formation of L568A/W571R and abolishes its potent antiviral activity.
(A) CD spectrum of L568A/W571R/I573S at 0°C in PBS (pH 7.0) at a
peptide concentration of 10 µM. The inset shows a thermal melt
monitored by CD at 222 nm for L568A/W571R/I573S in PBS (pH 7.0) at a
peptide concentration of 10 µM. (B) Inhibition of
HIV-1IIIB-infected H9 cell-induced cell-cell fusion
(circles) and HIV-1IIIB-mediated CPE (triangles) by
L568A/W571R/I573S. Error bars indicate standard deviations from
quadruplicate and triplicate experiments for cell-cell fusion and
infectivity assays, respectively.
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DISCUSSION |
Core structure and mechanism.
Recent descriptions of the gp41
ectodomain core structure provide detailed information about the
fusogenic conformation of the HIV-1 envelope protein (3, 10, 56,
57). The structure reveals a three-stranded coiled coil adjacent
to the N-terminal fusion peptide, surrounded by an outer layer of
antiparallel -helices. This structural organization is shared by the
influenza virus and Moloney murine leukemia virus transmembrane
envelope proteins (2, 22), suggesting conservation of a
common core structure. The overall fusion mechanism of these viral
envelope proteins is thought to involve a conformational change and
subsequent antiparallel association of -helices that lead to
membrane apposition and fusion (23, 27, 57). A similar
mechanism is also proposed for the SNARE proteins in the fusion of
vesicles with their target membranes (52, 55).
Although the functional role of the gp41 core in promoting membrane
fusion remains largely undefined, additional information
has come from
the functional analysis of engineered HIV-1 envelope
protein variants.
Mutagenesis studies demonstrate that residues
that stabilize this core
structure are critical for membrane fusion
activity (
5,
12,
18,
58). Moreover, the folding and stability
of the N34(L6)C28 model
in vitro correlate well with severity
of the in vivo phenotypes
observed in cells expressing envelope
proteins bearing mutations at Ile
573 (
18,
40). Thus, the
gp41 core structure appears to play
a direct role in membrane
fusion. Interestingly, point mutations in the
Leu 568 and Trp
571 residues lining the right wall of the conserved
hydrophobic
cavity in the coiled coil of gp41 result in mutant envelope
proteins
that are completely defective in mediating membrane fusion
(
5).
Our results indicate that the N34(L6)C28 core structure
is destabilized
by the fusion-defective mutations. Remarkably, our
results also
indicate clearly that these substitutions do not prevent
the six-helix
bundle formation. The crystal structures of the wild-type
and
mutant molecules can be superimposed, with rms deviations of 0.67
Å for L568A and 0.45 Å for W571R. From the structures we can see
that
each mutant sequence is accommodated in the six-helix bundle,
resulting
in hydrophobic cavities that are surrounded by different
sets of atoms.
Taken together, these data suggest that a protein-folding
defect in the
gp41 core is unlikely to be a major cause of the
fusion-defective
phenotype of the mutant viruses. We propose that
the conserved
hydrophobic cavity within the fusogenic gp41 core
plays a specific and
key role during the membrane fusion step
of HIV-1
infection.
Inhibition of HIV-1 infectivity.
Synthetic N- and C-terminal
peptides of gp41 inhibit HIV-1 infection and syncytium formation
(30, 60, 62). Several considerations provide good evidence
that these peptides act by binding to virus gp41 and preventing the
fusogenic gp41 core formation (9, 11, 23, 34, 41, 45, 49,
61). First, inhibition of syncytium formation by the C43 peptide
is markedly reduced when stoichiometric amounts of the N51 peptide are
also present (41). Moreover, when a gp41 ectodomain
maltose-binding chimeric protein contains a proline mutation in the
trimeric coiled-coil region of gp41, the resulting mutant is a potent
inhibitor of HIV-1 infection (11). Second, the inhibitory
activity of a C-terminal peptide depends on its ability to interact
with the N-terminal coiled coil (9, 61). Third, the
structural features of the gp41 core are fully consistent with the
simple dominant-negative model of inhibition (10, 41, 56,
57). The C-terminal peptides block membrane fusion by binding to
the coiled coil of gp41, while the N-terminal peptides act as
inhibitors by preventing the trimeric coiled-coil formation and/or
associating with the endogenous C-terminal region of gp41. Finally,
kinetic studies suggest that these peptides do not act on the native
conformation of the HIV-1 envelope (23) but can act during
its transition to the fusogenic state (23, 32, 45).
The use of the single-chain N34(L6)C28 model for the study of the gp41
core structure leads to the interesting result that
N34(L6)C28 also
inhibits HIV-1 infectivity. We have defined the
following
characteristics of this inhibitory activity. First,
the inhibition is
not attributable to the isolated N34 and C28
peptides, because the
N34(L6)C28 trimer is still highly stable
to thermal denaturation at its
IC
50, with an apparent
Tm of
approximately
63°C (
40). Second, there is amino acid
sequence-specific inhibition
by N34(L6)C28. The inhibitory activity is
dramatically increased
by the fusion-defective Leu 568-to-Ala and Trp
571-to-Arg mutations;
W571R, L568A, and L568A/W571R exhibit 5-, 16-, and 35-fold greater
activity than the wild-type molecule, respectively.
Third, the
inhibition is also conformation specific; the presence of
the
Ile 573-to-Ser mutation in the double mutant essentially disrupts
the six-helix bundle formation, while reducing its inhibitory
activity
16-fold. Fourth, the enhanced inhibitory activity by
these mutations
correlates with local structural perturbations
near the hydrophobic
cavity which destabilize the N34(L6)C28 trimer.
Further studies of the
inhibition mechanism of the gp41 core should
provide insights into the
HIV-1 entry process and could open new
perspectives in the search for
effective antiviral
therapies.
Implications for membrane fusion.
The hemagglutinin protein of
influenza virus irreversibly switches from the native structure to the
fusogenic conformation when exposed to the acidic environment of the
cellular endosome (2, 6, 26, 63). This structural dimorphism
is the basis for conformational changes that are crucial for activation
of membrane fusion. The HIV-1 envelope protein is also thought to exist
in two different conformations (for recent reviews, see references
8 and 52). It is generally
accepted that the native conformation exists on the surface of free
virions, while upon binding of gp120 to CD4 and particular coreceptors
(e.g., CCR5 or CXCR4), the HIV-1 envelope protein undergoes a complex
of structural changes to the fusogenic state. The current model for
gp41-mediated membrane fusion suggests that formation of the six-helix
bundle leads to colocalization of the viral and cellular membranes for fusion (23, 27, 57). While relatively little is known about how membrane apposition leads to complete fusion, there is evidence for
the higher-order assembly of envelope protein trimers and the formation
of fusion pores, as proposed to be required for influenza virus fusion
(20, 53, 59).
Since the gp41 ectodomain core structure, with a
Tm in excess of 90°C, is too stable to be
disrupted by exogenous peptide
binding, only during the gp41
conformational change to the fusogenic
state does one anticipate that
the targets for the peptides are
available (
11,
23,
34,
40,
41). This consideration has
led to the proposal that gp41 can
exist as a transiently populated
intermediate after initiating the
receptor-activated conformational
change but prior to formation of the
six-helix bundle (
8,
23,
45). According to this view,
synthetic peptides derived from
the gp41 ectodomain inhibit membrane
fusion in a dominant-negative
manner by associating with their
endogenous partners of viral
gp41 at this intermediate
stage.
Earlier genetic studies indicate that mutations in the Leu 568 and Trp
571 residues abolish membrane fusion activity, although
the mutant
HIV-1 envelope proteins appear to have no other defects,
including cell
surface expression, gp160 precursor processing,
and CD4 binding
(
5). Our results indicate that these fusion-defective
mutations destabilize the gp41 core structure although they still
confer the six-helix bundle fold. Since the Leu 568 and Trp 571
residues form the right wall of a conserved coiled-coil cavity
that
provides a binding pocket for three C-terminal helices (
9),
our data suggest that the fusion-defective mutations introduce
structural perturbations in the cavity that weaken helical packing
interactions in the six-helix complex and thus inhibit its
formation.
These fusion-defective mutations also exert striking effects on the
inhibitory activity of N34(L6)C28; the L568A and W571R
mutants exhibit
5- to 16-fold-greater activity than the wild-type
molecule. Several
lines of evidence suggest that this enhanced
inhibitory activity
results from the synergistic inhibition of
the N34 and C28 peptides in
the mutant molecules. First, while
the L568A and W571R trimers are
stable, with
Tm values of 56 and
61°C,
respectively, in PBS (pH 7.0) at a peptide concentration
of 10 µM,
L568A and W571R are predominantly unfolded at their
IC
50s
(0.1 µM for L568A and 0.3 µM for W571R) under physiological
conditions. The monomeric forms of the L568A and W571R molecules
readily interact bivalently with virus gp41. Second, the Ile 573-to-Ser
mutation that disrupts the N34 coiled-coil formation (
40)
can
reduce the potency of the double mutant (L568A/W571R) in inhibiting
membrane fusion close to that of the isolated C28 peptide. The
nature
of the multivalency in the N34(L6)C28 variants is likely
to be
responsible for their enhanced inhibitory activity. Finally,
this
synergy is fully consistent with the hypothesis that there
is a
populated intermediate of gp41 during transition to the fusogenic
structure (
8,
23,
45). Only in the intermediate state are
the N- and C-terminal heptad-repeat regions of virus gp41 not
associated, allowing the N34 and C28 peptides to bind to these
regions
with a high effective
concentration.
| |
ACKNOWLEDGMENTS |
We thank Jun Dong for suggestions on structural refinement and
Neville Kallenbach for critical reading of the manuscript.
This research was funded by NIH grants (AI-42693 to S.J. and AI-42382
to M.L.) and by the New York City Council Speaker's Fund for
Biomedical Research (to M.L.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Phone: (212) 746-6562. Fax: (212) 746-8875. E-mail: mlu{at}mail.med.cornell.edu.
| |
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Abstract
Introduction
