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Journal of Virology, December 1998, p. 9676-9682, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutational Analysis of Residues in the Coiled-Coil Domain of
Human Immunodeficiency Virus Type 1 Transmembrane Protein
gp41
Yongkai
Weng and
Carol D.
Weiss*
Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, Maryland 20892
Received 25 June 1998/Accepted 20 August 1998
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ABSTRACT |
The envelope glycoprotein (Env) of human immunodeficiency virus
mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A
six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and
P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen,
S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature
387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu,
Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects
of amino acid substitutions of arginine or glutamic acid in residues in
the coiled-coil (heptad repeat) domain that line the interface between
the helices in the gp41 core structure. We found that mutations of
leucine to arginine or glutamic acid in position 556 and of alanine to
arginine in position 558 resulted in undetectable
levels of Env expression. Seven other mutations in six positions
completely abolished fusion activity despite incorporation of the
mutant Env into virions and normal gp160 processing.
Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied,
although both viable mutants exhibited impaired fusion activity
compared to that of the wild type. The glutamic acid 577 mutant was
more sensitive than the wild type to inhibition by a gp41
coiled-coil peptide (DP-107) but not to that by another peptide
corresponding to the C helix in the gp41 core structure (DP-178).
These results provide insight into the gp41 fusion mechanism and
suggest that the DP-107 peptide may inhibit fusion by binding to the
homologous region in gp41, probably by forming a peptide-gp41
coiled-coil structure.
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INTRODUCTION |
The envelope glycoprotein (Env) of
human immunodeficiency virus (HIV) attaches virus to target cells and
fuses viral and cellular membranes, releasing the viral nucleocapsid to
the cell cytoplasm (reviewed in reference 14). Env
is synthesized as a fusion-inactive precursor (gp160) that is cleaved
in the biosynthetic pathway to generate the mature, noncovalently
associated surface (gp120) and transmembrane (gp41) subunits
(21). gp120 binds to the CD4 cellular and chemokine
receptors (13, 23, 25), and gp41 catalyzes membrane fusion
(18, 19). Cleavage of gp160 positions a hydrophobic
stretch of residues (fusion peptide) at the extreme N
terminus of gp41 (4, 16) that is believed to initiate fusion by directly inserting into the target cell membrane.
The mechanism of Env-mediated membrane fusion is not known, but
precedents set by other viruses suggest that the fusion process involves large conformational changes in Env and movement of Env oligomers in the plane of the membrane (reviewed in reference 3). The best-characterized viral fusion
glycoprotein, the hemagglutinin of influenza virus, undergoes dramatic
conformational changes when the virus is exposed to mildly acidic pH
during receptor-mediated endocytosis (reviewed in reference
36). A critical part of the conformational change
involves a transition from a loop to a helix structure in a heptad
repeat segment near the fusion peptide, creating an extended,
triple-stranded, coiled coil (reviewed in reference
20) that relocates the fusion peptide approximately 100 Å closer to the target cell membrane (the "spring-loaded
mechanism") (5, 7).
Several studies have indicated that conformational changes occur when
Env binds CD4 (26, 29, 30). Recently, it has been proposed
that Env binding to receptor triggers conformational changes in a
coiled-coil (heptad repeat) region in gp41 near the fusion peptide
(17). At present, it is not clear whether this change
involves altered exposure of the coiled-coil region or a change
in conformation such as a loop-to-helix transition as is the case for
hemagglutinin activation. The coiled-coil segment probably
subsequently associates with a downstream gp41 amphipathic helical segment to form an extremely stable alpha-helical bundle, consisting of an internal, triple-stranded parallel coiled-coil core
(heptad repeat segment) with three antiparallel gp41
amphipathic alpha helices packing in the grooves (see Fig. 1B, core
structure), as shown in crystallographic studies of gp41 peptides that
self-assemble (8, 32, 35).
In this report, we describe the results of studies involving mutations
in the coiled-coil domain (residues 553 to 595, hereafter referred to
as the heptad repeat) near the fusion peptide of gp41 that potentially
affect both the stability of the coiled-coil core itself and the
interactions with the external antiparallel helices (see Fig. 1A and
B). The mutated residues occupy the "e" and "g" positions in
the heptad repeat motif (abcdefg) and generally line the groove of the
coiled-coil core against which the antiparallel outer helix packs (see
Fig. 1C). Several of these mutations also surround a deep hydrophobic
cavity that has been proposed as a good target for pharmacological
intervention (8). Our studies showed that 14 of the 16 nonconservative mutations in these residues completely abolish fusion
activity and that some mutations clustering in one region of the heptad
repeat inhibit Env expression. We further assessed the sensitivity of
each mutant to gp41 peptide inhibitors predicted to interact with this
region and discuss results in the context of structural requirements
for Env-mediated membrane fusion.
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MATERIALS AND METHODS |
Cells and plasmids.
293T, U87-T4-CXCR4, and U87-T4-CCR5
cells and the Env expression plasmid pSM-WT (HXB2) and Env-deficient
HIV reporter plasmid pNL43-Luc-R
E were
kindly provided by Dan Littman (New York University, New York, N.Y.).
COS-7 cells were obtained from the American Type Culture Collection
(Manassas, Va.). The REV expression vector pREV was kindly provided
by Tris Parslow (University of California, San Francisco). Mutant Envs
were generated by site-directed mutagenesis from a uridine-substituted
single-stranded template (pSM-WT) with the Bio-Rad mutagenesis kit
(Bio-Rad Laboratories, Hercules, Calif.). Primers used for mutagenesis
are available on request. Mutations were confirmed by sequencing with
the Sequenase quick-denature plasmid sequencing kit (U.S. Biochemical,
Cleveland, Ohio).
Envelope glycoprotein expression.
Approximately 2.5 × 105 COS-7 or 8 × 105 293T cells were
plated in six-well dishes (Falcon, Franklin Lakes, N.J.) 1 day prior to
transfection so that cells would reach 60 to 70% confluency on the day
of transfection. Two micrograms of Env plasmid DNA and 1 µg of pRev
DNA were cotransfected with the Lipofectamine reagent (GIBCO BRL,
Gaithersburg, Md.). After 6 h of incubation at 37°C, Dulbecco's
minimal essential medium (DMEM) containing 10% fetal calf serum (FCS)
was added to the cultures. The cultures were continued at 37°C for
40 h before harvesting for cell-cell fusion assays or immunoblots,
as described below.
Env expression in transfected cells was assessed by lysing cells with
0.1 ml of 1% Nonidet P-40 (NP-40)-150 mM NaCl-100 mM Tris (pH 8.0)
buffer (lysis buffer). Approximately 10 µl of the clarified lysate
was separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (4 to 12% NuPAGE gels; NOVEX, San Diego,
Calif.) and transferred to an ECL nitrocellulose membrane (Amersham,
Arlington Heights, Ill.). The membranes were then probed with
anti-gp120 polyclonal goat serum (Env 2-3; kindly provided by Kathelyn
Steimer, Chiron Corp., Emeryville, Calif.) at 1:5,000 in 5%
milk-phosphate-buffered saline, washed, reprobed with
peroxidase-conjugated anti-goat antiserum (Sigma, St. Louis, Mo.), and
then washed again before detection by chemiluminescence (Amersham) and autoradiography.
Cell-cell fusion assay.
COS-7 cells transfected with mutant
or wild-type Envs were assessed for fusion activity as previously
described (33). Briefly, approximately 5 × 106 SupT-1 lymphocytes were labeled with 6 µg of
calcein-AM (Molecular Probes, Eugene, Oreg.) for 30 min and then washed
before cocultivation with an equal number of adherent Env-expressing
cells. Fusion was scored approximately 1 h after cocultivation by
assessing dye transfer to Env-expressing cells under epifluorescence microscopy.
Pseudovirion generation and infectivity assay.
Pseudovirion
stocks with wild-type or mutant Envs were generated as previously
described (12). Briefly, 3 × 106 293T
cells in 10-cm dishes were cotransfected with 5 µg each of the Env
expression vector and the Env-deficient viral reporter vector
(pNL43-Luc-RE) by the Lipofectamine method
(GIBCO BRL). The supernatants containing pseudoviruses were
collected 48 h posttransfection and frozen at 
80°C. Virus
quantities were determined by p24 enzyme-linked immunosorbent assay
(Coulter, Westbrook, Maine). One day before infection, 3.5 × 104 U87-CD4-CXCR4 cells were plated in 48-well dishes
in DMEM containing 10% FCS, 2 mM glutamine, 1× antibiotics, 1 mM
sodium pyruvate, and 1× nonessential amino acids
(DMEM+) and infected with pseudovirus (80 ng of
p24) in a total volume of 300 µl containing 8 µg of Polybrene
(Sigma) per ml. After 8 h of incubation, 0.5 ml of fresh
DMEM+ containing 15% FCS was replaced in the wells, and
cultures were continued for 40 h at 37°C. The cells were then
lysed, and luciferase activity was measured with the luciferase assay
kit (Promega, Madison, Wis.) and a LumiCount (Packard, Meriden, Conn.) luminometer.
Detection of envelope glycoproteins in
pseudovirions.
gp120 in the pseudovirion
stocks was assessed by immunoprecipitating 250 ng of p24
pseudovirion stocks that were lysed with lysis buffer with
1 µg of soluble CD4-immunoglobulin G (IgG) (kindly provided by
Genentech, South San Francisco, Calif.) while mixing them at room
temperature for 1 h. Approximately 25 µl of a 25% suspension of
protein A-agarose (GIBCO BRL) was then added to the sample, and the
mixture was incubated for an additional hour at room temperature with
mixing. The precipitates were then washed three times with lysis
buffer, separated by SDS-PAGE (4 to 12% NuPAGE gels), and
immunoblotted with the polyclonal gp120 antiserum, as described above.
To detect gp41 in pseudovirions, pseudovirion
stocks containing 500 ng of p24 were centrifuged at 20,000 ×
g for 3 h at 4°C
to pellet pseudovirions.
Pseudovirion pellets were then lysed
with 50 µl of lysis buffer,
and half of the pseudovirion lysate
was applied to SDS-PAGE
gels (10% NuPAGE gels), immunoblotted
with Chessie 8 antibody
(
1) (obtained from National Institutes
of Health AIDS
Research and Reference Reagent Program), and detected
by
chemiluminescence and autoradiography, as described
above.
To detect soluble and pseudovirion-bound gp120 in the
pseudovirion stocks, pseudovirion stocks
containing 500 ng of p24 were
centrifuged at 20,000 ×
g for 3 h at 4°C to pellet pseudovirions.
Supernatants were adjusted to 1% (vol/vol) NP-40, and
pseudovirion
pellets were lysed with NP-40 lysis buffer.
The supernatant and
pseudovirion fractions were then
adjusted to equal volumes with
lysis buffer before immunoprecipitation
with soluble CD4-IgG,
separation by SDS-PAGE (10% NuPAGE gels), and
immunoblotting with
gp120, as described above. Detection for these
blots involved
alkaline phosphatase-conjugated secondary antibody and
development
with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate
substrate
(Promega).
Peptide inhibition assay.
The peptides DP-107
(NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ) and DP-178
(YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF) (kindly provided by Carl
Wild, Biotech Research Laboratories, Gaithersburg, Md.) were
synthesized by 9-fluorenylmethoxycarbonyl chemistry, purified by
high-performance liquid chromatography on a C18 column, and analyzed by electron spray mass spectroscopy. Then 3.5 × 104 U87-T4-CXCR4 cells per well were plated in 48-well
dishes in DMEM+ 1 day before infection and inoculated with
80 ng of p24 pseudovirus containing peptide at the
indicated concentration in a total volume of 300 µl of
DMEM+ with 8 µg of Polybrene per ml. After 8 h of
incubation, the inoculum containing peptide was replaced with 0.5 ml of
fresh DMEM+ containing 15% FCS, and cultures were
continued for 40 h at 37°C. Infection was determined by the
luciferase assay as described above.
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RESULTS |
Effect of gp41 mutations on Env expression.
Heptad repeat
motifs, characteristic of coiled coils, typically contain bulky
hydrophobic residues that alternate every third and fourth residue (the
"a" and "d" positions) to create a hydrophobic face to an alpha
helix (Fig. 1A), (reviewed in reference
2). Residues in the "a" and "d" positions in
opposing helices interact hydrophobically to stabilize the coiled-coil
structure (Fig. 1C). In addition, ionic interactions in the
"e" and "g" positions can influence coiled-coil specificity and
have been shown to influence heterodimerization versus homodimerization
of coiled-coils, as in the case of the Fos and Jun transcription
factors (27). Previous mutational studies of the gp41 heptad
repeat segment have shown that mutations in the "a" and "d"
positions, particularly helix-disrupting mutations, impair fusion
activity (6, 10, 11, 15). In this study, we introduced
mutations in the "e" and "g" positions because these residues
occupy positions that can potentially interact with the external
antiparallel helices as well as helices in the coiled coil itself.
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FIG. 1.
(A) Map of gp41 showing heptad repeat residues,
mutations, and helical regions and peptides that make up the core
structure. (B) Schematic diagram of six-helix bundle of the gp41 core
structure, showing overlap with the DP-107 and DP-178 peptides. (C)
Cross section through core structure showing position of heptad repeat
residues in the coiled-coil domain. TM is the transmembrane region of
gp41. The breaks in the cytoplasmic domain indicate that this region is
longer than what is shown. Shaded cylinders represent the coiled-coil
domain (N helices), which overlaps the DP-107 peptide. White cylinders
represent the external helices (C helices), which overlap the DP-178
peptide.
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Eight "e" and "g" positions from residues 556 to 588 (HXB2
strain) in the heptad repeat region were mutated to arginine or
glutamic acid (Fig.
1A). These nonconservative substitutions were
chosen because these amino acids occur naturally in other "e"
and
"g" positions in the heptad repeat segment and are unlikely
to
disrupt overall helix structure. In addition, we hoped that
these
charged residues would facilitate analysis of gp41 peptide
inhibitors
that are predicted to interact with this region. For
the position
572 mutant, alanine was substituted instead of arginine
or
glutamic acid because the naturally occurring glycine, which
is an
uncommon residue in helical structures, suggested that only
a
small amino acid would be
tolerated.
Studies examining transient Env expression in whole-cell lysates showed
that both arginine and glutamic acid substitutions
in the 556 position
(556R and 556E, respectively) resulted in
undetectable amounts of Env
expression (Fig.
2A and Table
1).
An alanine was also substituted in
this position (556A) to assess
whether a more conservative mutation
would be tolerated. This
mutant expressed Env but showed impaired
cleavage of the gp160
precursor relative to that for wild type.
Arginine substitution
at the 558 position (558R) also resulted in
undetectable amounts
of Env, but the double mutant with an arginine
substitution in
both the 556 and 558 positions (556/58R) restored Env
cleavage
and expression, though at reduced levels. Glutamic acid
substitution
in position 558 (558E) resulted in expression of a
faster-migrating
Env with reduced expression levels. All other single
mutants (570E,
570R, 577R, 577E, 586E, and 586R) and two double mutants
(556/58R
and 577/84R) expressed substantial amounts of Env and showed
normal
gp160 cleavage. One double mutant with glutamic acid
substitutions
in positions 577 and 579 (577/79E) showed greatly
reduced Env
expression compared to that of wild type.
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FIG. 2.
(A) Immunoblots of cell lysates transiently expressing
wild-type or mutant Envs probed with a polyclonal anti-gp120 antiserum.
(B) Immunoblots of wild-type or mutant Env pseudovirion
stock lysates probed with a polyclonal anti-gp120 antiserum after
immunoprecipitation with CD4-IgG. env() is a negative control using
REV expression vector only (A) or Env-deficient HIV reporter vector
(B). pSM-WT is the wild-type Env. Control is a cell lysate from stable
CHO cells expressing Env. Arrows indicate gp120 and gp160 bands. The
asterisk indicates IgG heavy chain. Experiments were performed at least
three times with similar results.
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We also assessed Env expression in the context of virion production, by
generating pseudovirions with the mutant and wild-type
Envs
with an Env-defective, HIV packaging vector (
9) (Fig.
2B).
gp120 present in these pseudovirion stocks likely
represents
a mixture of gp120 shed from the cell surface, gp120
secreted
in the biosynthetic pathway (
31), and gp120
incorporated into
pseudovirions. Immunoprecipitation
studies of these pseudovirion
stocks with CD4-IgG showed
that total Env expression in the pseudovirion
stocks
paralleled overall Env expression in cell lysates with
the Env
expression vector alone (Fig.
2A). The greater amounts
of gp120 than of
gp160 in the pseudovirion stocks compared to
cell lysates
probably reflect several factors, including a high
degree of gp120
shedding seen with the HXB2 strain, better binding
of gp120 than of
gp160 to CD4-IgG (data not shown), and possibly
also better
incorporation of the cleaved mature Env than of the
gp160 precursor
into the
pseudovirions.
Effect of gp41 mutations on infectivity.
The effects of the
gp41 mutations on HIV entry were assessed in a single-round,
infectivity assay using pseudotyped viruses generated from the mutant
Envs and an Env-deficient HIV packaging vector containing a luciferase
reporter gene (12). Our results showed that most positions
appeared to be intolerant of nonconservative changes as demonstrated by
complete inhibition of infectivity with two different mutations (Fig.
3). Only the 570E and 577E mutants
retained significant infectivity. Both of these mutants were impaired
in infectivity, with the 570E and 577E mutants showing approximately 76 and 18% fusion activity, respectively, compared to that of wild
type. In contrast to these glutamic acid mutants, substitution of
arginine in these same positions caused complete loss of infectivity.
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FIG. 3.
Infectivity of wild-type and mutant Env pseudotyped
viruses. y axis shows luciferase activity as a measure of
infectivity as described in Materials and Methods. Luciferase activity
is shown as relative luciferase activity units normalized to wild type.
Data points are averages of three independent experiments. Error bars
show 1 standard deviation each and are absent where standard deviations
are too small to show.
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Because the single-round infectivity assay depends on virus uncoating,
reverse transcription, integration, and transcription
and translation
of a reporter gene, in addition to Env function
to measure infectivity,
the effects of the mutation on Env fusion
activity were confirmed in a
virus-free cell-cell fusion assay
(Table
1). The results of these
studies paralleled those of the
infectivity assay, showing that only
570E and 577E had fusion
activity.
Effect of gp41 mutations on Env incorporation into virions.
With the exception of the single mutants involving residues in the 556 and 558 positions and the double mutants 556/58E and 577/79E, most
mutants demonstrated Env expression in pseudovirion stocks
(Fig. 2B), and yet only two mutants (570E and 577E) produced infectious
virus. In order to determine more directly whether the gp120 detected
in the pseudovirion stocks accurately reflected incorporation of the mutant Envs into the pseudovirions, we
pelleted the pseudovirions and probed the
pseudovirion lysates for gp41 (Fig.
4A and Table 1). For these studies, we
analyzed only the mutants that showed good Env expression in earlier
studies. Immunoblots of the pseudovirion lysates showed
gp41 bands in all samples, but the levels for 556/58R and 572A mutants
were reduced well below those for wild type and the other mutants. The
reduced detection of gp41 in the 572A mutant is consistent with the
inefficient cleavage of gp160 seen in previous experiments.
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FIG. 4.
Incorporation of wild-type and mutant Envs into
pseudovirions. (A) Immunoblots of pseudovirion
lysates probed with anti-gp41 monoclonal antiserum. Experiments were
performed twice with similar results. Arrows indicate gp160 and gp41
bands. (B and C) Pseudovirion stocks were centrifuged to pellet virus,
and the pellet (p) or supernatant (s) fractions were then
immunoprecipitated with CD4-IgG and immunoblotted with polyclonal
anti-gp120 antiserum. Control is a cell lysate from stable CHO cells
expressing Env. Arrows indicate gp120 and gp160 bands. These
experiments were performed three times with similar results. WT, wild
type.
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Pseudovirion lysates and supernatants from pelleted
pseudovirion stocks were also assessed for gp120 and gp160
by immunoprecipitation
with CD4-IgG (Fig.
4B and C). All mutants
showed gp120 bands in
both pellet and supernatant fractions, confirming
incorporation
of the mutant Envs into the pseudovirions. We
noted better detection
of gp120 than of gp41 in the pellet fractions
for the 556/58R
mutant and especially the 572A mutant, but the reasons
for this
are not clear. Consistent with the high degree of gp120
shedding
with the HXB2 strain, both wild-type and mutant Env
pseudovirion
stocks showed stronger gp120 detection in the
supernatant fraction.
Although the relative degree of gp120 shedding
between the mutants
and wild type was not rigorously quantified, it
appears unlikely
that the differences could account for the loss of Env
function
seen in the
mutants.
Effect of gp41 mutations on sensitivity to peptide inhibition.
Two peptides (DP-107 and DP-178) corresponding to helical regions in
gp41 have been shown elsewhere to be potent inhibitors of HIV
infectivity (37, 39, 40) (Fig. 1A). These peptides overlap
the gp41 helices that self-assemble to form the gp41 core structure
(Fig. 1B). It is believed that the gp41 inhibitory peptides bind
gp41 and in a dominant-negative manner prevent the formation of the Env
structure(s) required for fusion (22, 24). Recently, it has
been shown that the DP-178 peptide directly binds gp41 after gp120
binds receptors, indicating that the heptad repeat domain (DP-107
region) changes conformation and/or becomes accessible during fusion
and that changes in the gp41 core structure occur during entry
(17). We therefore tested the DP-107 and DP-178 peptides
against fusion-competent gp41 mutants.
The dose-response curves of the DP-178 peptide against
wild-type or mutant viruses did not show significant
differences in
sensitivity to peptide (Fig.
4A). In contrast, the 577E
mutant
virus appeared to be more sensitive to inhibition by the DP-107
peptide, giving 50% inhibition at fourfold-lower concentrations
than
those that are required to inhibit wild-type and 570E viruses
(Fig.
4B). This increased sensitivity to inhibition by the DP-107
peptide is
probably specific to the 577E mutation and not a consequence
of
impaired fusion activity, because the 577E mutant can be inhibited
by
the DP-178 peptide, similar to wild type. This finding suggests
that
the 577E mutation facilitates binding of the DP-107 peptide
to
gp41.
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DISCUSSION |
The heptad repeat segment of gp41 is one of the most highly
conserved regions in Env and plays a critical role in HIV entry. In
this report, we analyzed the effects of substitution in residues in the
heptad repeat that generally line the grooves between the coiled-coil
core and the exterior antiparallel helices (Fig. 1C). Mutations in
these "e" and "g" positions potentially affect the stability of
the coiled coil itself as well as interactions with the external helix
to form the core structure. We found that nonconservative mutations at
two positions (556 and 558) near the N terminus of the heptad repeat
(DP-107) region resulted in undetectable levels of Env expression.
These results may be due to misfolding of Env and rapid degradation.
However, because the mutations also lie in the Rev-responsive
element, impaired expression could relate to problems of mRNA transport
from nucleus to cytoplasm. The extreme conservation of the heptad
repeat region may be due to the combined selective pressure for Env and
Rev function.
Eight nonconservative mutations (570E, 570R, 577E, 577R, 586E, 586R,
556/58R, and 577/84R) generated Envs that were incorporated into
virions, processed to gp120, and able to bind CD4, indicating that
these mutations probably did not cause major disruption of the Env
structure in its native conformation (prior to fusion-inducing changes). Yet only the 570E and 577E mutants were fusion competent. Both of these mutants were impaired relative to wild type; the 570E and
577E mutant Envs showed infectivity at approximately 76 and 18% of
wild-type levels, respectively. Although several fusion-incompetent
mutants showed reductions in virion-associated Env compared to wild
type (577E, 577/84R, and 556/58R), these differences are unlikely to
account for the complete absence of fusion function. Therefore, the
mutations in the eight fusion-incompetent mutants that showed gp120 and
gp41 incorporation in virions likely affect a fusion-active
conformation of Env, possibly the core structure. Interestingly, the
570 and 577 residues occupy "e" positions one helical turn away
from each other and lie on the same face of the deep cavity shown in
the crystallized core structure. For both the 570 and 577 positions,
glutamic acid but not arginine substitutions permitted Env function. A
neutral mutation from glycine to alanine in position 572 also
completely abolished fusion activity.
Experiments comparing inhibition of mutant and wild-type viruses by the
DP-107 and DP-178 peptides showed differences only with the DP-107
peptide against the 577E virus. The crystal structures of the gp41
core domain indicate that the 570 and 577 positions in the coiled-coil
domain are distant from residues corresponding to the DP-178 peptide.
Therefore, it is not surprising that inhibition by the DP-178 peptide
was not significantly different between the 570E and 577E mutants
compared to wild type. The crystallography data also indicate that
glutamine 577 has potential for hydrogen bonding with tryptophan 628 of
the exterior helix (Fig. 5). Perhaps loss
of hydrogen bonding between these residues by the glutamic acid
substitution at position 577 contributes to impairment of fusion
activity, through destabilization of the interactions between the
external helix and the coiled-coil core.
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FIG. 5.
Dose-response curves of inhibition by the DP-178 (A) and
DP-107 (B) peptides against the wild-type (WT), 570E, and 577E Env
pseudotyped viruses. y axis shows luciferase activity as a
measure of infectivity as described in Materials and Methods. Data
points are averages of three independent experiments. Error bars show 1 standard deviation each and are absent where standard deviations are
too small to show.
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The crystal structure also suggests ways that the 577E mutant could be
more sensitive to inhibition by the DP-107 peptide. Molecular modeling
of the 577E mutant using coordinates from the core crystal
structure (34) (Insight software; Molecular
Simulations Inc., San Diego, Calif.) shows the potential for ionic
interactions between glutamic acid 577 in gp41 and arginine 579 in the
neighboring coiled-coil helix, representing interactions between
"e" and "g" residues in opposing coiled-coil helices (Fig.
6). Conceivably, this ionic interaction
may improve binding of the DP-107 peptide to the mutant 577E Env,
resulting in greater inhibition of function compared to wild-type Env,
which has no potential for ionic interactions with the DP-107 peptide
in this position. Molecular modeling of the 570E mutant did not show
similar potential for improved binding to the DP-107 peptide. Because
data from peptide studies demonstrate that DP-107 and related peptides
interact with themselves to form trimeric coiled coils and also
interact with DP-178 and related peptides to form heterodimers (8,
22, 28, 32, 34, 38), the DP-107 peptide could block HIV
entry by binding to the DP-107 region and/or the DP-178 region in
gp41. The increased sensitivity of the 577E mutant (in the DP-107
region) to inhibition by the DP-107 peptide (wild-type sequence)
suggests that the DP-107 peptide may work by binding to the homologous
region of gp41, preventing formation of the gp41 coiled-coil structure
required for fusion.
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FIG. 6.
Helical wheel representation of cross section through
core structure, showing residues in the "e" and "g" position in
the coiled-coil domain and neighboring residues in the external helix.
Numbers indicate potential interacting residues (numbering for HXB2
strain). Changing glutamine in position 577 to glutamic acid destroys
potential hydrogen bonding with the tryptophan in position 628 and
creates potential ionic interaction with arginine in position 579.
|
|
These data provide insights into the structural constraints in the gp41
fusion mechanism and also suggest that the heptad repeat region would
be a good target for therapeutic intervention. Our data indicate that
mutations in this position are often lethal to the virus, so the
ability of HIV to mutate and become resistant to inhibitors directed
against the heptad repeat region is likely to be very limited.
| |
ACKNOWLEDGMENTS |
We thank Ira Berkower and Darón Freedberg (Center
for Biologics, Food and Drug Administration, Bethesda, Md.) for
critical review of the manuscript, Rika Furuta (Center for Biologics,
Food and Drug Administration) and Carl Wild (Biotech Research
Laboratories) for many helpful discussions, and Terry Oas (Duke
University, Durham, N.C.) for molecular modeling of the 570 and
577 mutants.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: FDA/CBER,
HFM-413, Bldg. 29, Room 532, 29 Lincoln Dr., Bethesda, MD 20892-4555. Phone: (301) 402-3190. Fax: (301) 496-4684. E-mail:
cdweiss{at}helix.nih.gov.
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Journal of Virology, December 1998, p. 9676-9682, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
