JOURNAL OF VIROLOGY, Mar. 2004, p. 2627–2631
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 5
Binding of the 2F5 Monoclonal Antibody to Native and
Fusion-Intermediate Forms of Human Immunodeficiency
Virus Type 1 gp41: Implications for Fusion-Inducing
Eve de Rosny,1† Russell Vassell,1Shibo Jiang,2
Renate Kunert,3and Carol D. Weiss1*
Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland1;
Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York2;
and Institute of Applied Microbiology, Universita ¨t fu ¨r Bodenkultur, Vienna, Austria3
Received 7 August 2003/Accepted 11 November 2003
We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency
virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5
binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We
also demonstrate conformational changes in the C heptad of gp41.
The envelope glycoprotein (Env) of human immunodefi-
ciency virus type 1 (HIV-1) consists of a surface (gp120) sub-
unit that attaches virus to target cells and a noncovalently
associated transmembrane protein (gp41) (Fig. 1A) that me-
diates fusion between virus and target cell membranes. These
subunits are synthesized as a fusion-inactive precursor (gp160)
that is cleaved in the synthetic pathway to generate mature
Env. gp120 binding to CD4 and chemokine receptors on the
host cell triggers fusion-inducing conformational changes in
gp41, leading to entry of the viral nucleocapsid into the host
The mechanism of gp41-mediated membrane fusion is not
fully understood. A widely accepted model of HIV entry pos-
tulates that gp41 undergoes major refolding steps as it medi-
ates membrane fusion (Fig. 1B, reviewed in reference 5). In
this model, gp41 transitions from its native, metastable confor-
mation as it exists on the surface of virus or infected cells to a
final fusion-active conformation consisting of a thermostable
six-helix bundle structure (Fig. 1C). This structure forms when
two heptad repeat regions in the gp41 ectodomain self assem-
ble into a trimer of hairpins, where the N- and C-terminal
heptad repeats align in an antiparallel manner in each hairpin
monomer (4, 35, 37). The N heptads form a triple-stranded
coiled coil in the internal layer of the six-helix bundle, and the
C-heptad repeat helices form the external layer. It is believed
that gp120 binding to receptors loosens the association of
gp120 with gp41, which in turn releases the fusion peptide at
the N terminus of gp41 to insert into the target membrane.
Subsequent folding of this fusion-intermediate conformation
into the six-helix bundle structure probably facilitates fusion by
bringing membranes together as Env adopts a more thermo-
dynamically stable conformation.
We investigated gp41 conformational changes by analyzing
how two gp41 monoclonal antibodies (MAbs) with epitopes in
the C heptad of gp41 bind Env under various conditions (Fig.
1A). The first antibody, 2F5, well known for being one of the
few broadly neutralizing and protective HIV antibodies (20,
28), binds to a core epitope containing the residues ELDKWA
at the C terminus of the C heptad (24, 27). The second anti-
body, D50, also binds a linear peptide from the C heptad but
is not neutralizing (7). D50 was generated in mice immunized
with a secreted, uncleaved, oligomeric form of Env (8). In
enzyme-linked immunosorbent assay experiments (data not
shown), we confirmed that both MAbs bind the same C-heptad
gp41 peptide (DP-178/T20, residues 638 to 673 of the HXB2
Env) but not an overlapping C-heptad gp41 peptide (C34,
residues 628 to 661 of the HXB2 Env).
We first assessed MAb binding to native or receptor-trig-
gered Env from the cell or virion surface (Fig. 2). Approxi-
mately 107293T cells transiently expressing HXB2 Env were
suspended in 500 ?l of Dulbecco’s modified Eagle’s medium
(DMEM) and were preincubated for 1 h at 37°C in the pres-
ence or absence of 2 to 4 ?g of a soluble form of CD4 (sCD4;
kindly provided by Ray Sweet, SmithKline Beecham, King of
Prussia, Pa.) to enrich for fully triggered Env. We and others
have shown that CD4 is sufficient for triggering Env into the
six-helix bundle (6, 15). The transfection was performed by
using the pSM-HXB2 and pREV expression plasmids and FU-
GENE 6 (Roche, Indianapolis, Ind.) as previously described
(13). 2F5 (1 ?g) or D50 containing supernatant (50 ?l) was
then added to cells and incubated for an additional hour at
37°C before being washed twice with phosphate-buffered saline
to remove unbound antibodies. Cells were then lysed and im-
munoprecipitated with protein A-agarose beads (Roche) as
previously described (38). The same assay was performed on
HIV-1 pseudovirions (HXB2 Env) with approximately 1 ?g of
p24-containing pseudovirion stocks, prepared as previously de-
* Corresponding author. Mailing address: FDA/CBER HFM-466,
Bldg. 29, Room 532, 8800 Rockville Pike, Bethesda, MD 20892-4555.
Phone: (301) 402-3190. Fax: (301) 496-1810. E-mail: cdweiss@helix
† Present address: Laboratoire des proteines membranaires, Institut
de Biologie Structurale, F-38027 Grenoble Cedex 1, France.
scribed (38), except that unbound antibodies were eliminated
by centrifugation (2 h at 20,000 ? g) instead of washing.
These experiments showed that 2F5 preferentially binds na-
tive gp41 (prior to receptor activation) (Fig. 2A, lane 1) but
that D50 prefers the triggered form after receptor activation
(Fig. 2A, lane 4), consistent with some previous reports using
flow cytometry assays (25, 31). Similar results were seen with
intact virions (Fig. 2B). The close proximity of the two MAb
epitopes on the linear sequence of gp41 combined with the
opposite responses to pretreatment with sCD4 strongly argues
that this region of gp41 undergoes major conformational
changes after gp120 binds CD4. While the increased binding of
the D50 MAb after receptor activation could be solely due to
increased exposure of this region after gp120 triggering by
CD4, the decreased binding of the 2F5 MAb after receptor
activation makes this explanation less likely. Additionally, a
recent report by Barbato et al. involving structural studies of
gp41 peptides provides further support for conformational
changes in this region (1). It is possible that conformational
changes could involve interaction of the pretransmembrane
domain with the membrane (1, 19, 34), which could disrupt the
2F5 epitope, but our experiments do not directly address this
We then investigated binding of the MAbs to the gp41 fusion
intermediate. Receptor-activated gp41 was trapped with pep-
tides corresponding to the C-heptad repeat (C34, which lacks
the 2F5 core epitope) or the N-heptad repeat (N36) (Fig. 1A)
before immunoprecipitation with the MAbs. These peptides
are potent inhibitors of HIV entry (16, 21, 39, 40) and have
been shown to preferentially bind gp41 after receptor activa-
tion (11, 13, 18), mimicking interactions in the six-helix bundle.
By a dominant-negative mechanism, the C peptide binds the
N-heptad repeat of gp41 (Fig. 1B) and the N peptide binds the
C-heptad repeat of gp41, preventing formation of the six-helix
bundle using the endogenous heptad repeats from gp41 (re-
viewed in reference 5). One hour prior to immunoprecipitation
with D50 (50 ?l) or 2F5 (1.5 ?g), 500 ?l of 107CHO Env-
expressing cells (36) was preincubated with 2 to 4 ?g sCD4 and
10 ?g of either N36 or C34 inhibitory gp41 to trap the fusion
intermediate. Significantly, the loss of the 2F5 epitope is pre-
FIG. 1. (A) Linear representation of gp41 domains and approximate localization of epitopes for 2F5 and D50 monoclonal antibodies (MAbs).
Numbering is based on the HIV-1 HXB2 strain according to the Los Alamos National Laboratory database. N36 peptide contains the following
residues (546 to 581): SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL. C34 peptide contains the following residues (628 to 661):
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL. FP, fusion peptide; TM, transmembrane domain. (B) Model of HIV-1 entry involving
conformational changes in gp41. Brackets show a putative fusion intermediate that is trapped with the C-heptad repeat peptide, preventing
formation of the six-helix bundle. (C) Schematic representation of the six-helix bundle structure generated by self assembly of the N- and C-heptad
FIG. 2. CD4 dependence of antibody binding to gp41. 2F5 and D50
MAbs were used to immunoprecipitate gp41 on the surface of Env-
expressing cells (A) or virions (B) in the absence (?) or presence (?)
of sCD4. Blots were probed with an antibody to gp41. Control is an
immunoblot of Env-expressing cell lysate. Results shown are represen-
tative of at least three independent experiments.
2628 NOTESJ. VIROL.
vented when the fusion intermediate is trapped with peptides
(Fig. 3). This effect is more pronounced when the fusion in-
termediate is trapped with the C peptide (Fig. 3A, lane 4) than
it is with the N peptide (Fig. 3A, lane 6), despite apparently
comparable abilities of the N and C peptides to trap gp41 when
excess peptides are used (13).
The results with the trapped fusion intermediates show that
the 2F5 epitope remains present shortly after gp120 has been
triggered by CD4 but may be lost by the time the six-helix
bundle has fully formed. This finding further suggests that the
C-heptad repeat region of gp41 may need to interact with the
endogenous N heptad of gp41 in order to change to a confor-
mation that abolishes the 2F5 epitope. The hypothesis is con-
sistent with the observation that the C peptide, which binds the
N heptad of gp41 (13, 29), is better at preserving the 2F5
epitope in the triggered conformation than is the N peptide,
which binds the C heptad of gp41 (13, 30) (Fig. 3). Because the
N peptide probably binds the C-heptad repeat near the 2F5
epitope to make a six-helix bundle-like structure, the N peptide
may facilitate helix formation in the C heptad, and in doing so
may perturb the conformation of the 2F5 epitope. This possi-
bility was suggested in a previous study involving 2F5 binding
to gp41 peptides (12). Our demonstration that the 2F5 epitope
is present on the fusion intermediate agrees with two publica-
tions that used different assays (9, 10), but there are inconsis-
tencies in the literature on this point (1).
In contrast to 2F5, no significant changes were seen with
immunoprecipitations using D50 when Env was first triggered
and trapped with either peptide (Fig. 3B, lanes 1 to 6). Thus,
it seems that the D50 epitope is equally present in the fusion-
intermediate and six-helix bundle conformations, suggesting a
linear epitope. The finding that the C peptide does not in-
crease immunoprecipitation by D50 is also significant, because
it rules out the possibility that the increased immunoprecipi-
tation of gp41 by 2F5 is due to stabilization of gp41 oligomers
as a result of interactions with the C heptad.
Our data lead us to propose that the C-heptad/pretrans-
membrane region may not be in a complete helical structure in
the native conformation of Env. Instead, helicity may increase
as gp41 folds into the six-helix bundle, perhaps as the gp41 C
heptad interacts with its N heptad. Such a scenario is reminis-
cent of the spring-loaded mechanism of fusion activation for
influenza hemagglutinin (3). This hypothesis is also supported
by peptide (26) and recombinant protein (14) studies, suggest-
ing that the 2F5 epitope is in a ?-turn-like conformation. On
the other hand, other studies indicate that the 2F5 epitope is
present in helical peptides (2, 17, 32). Based on these peptide
models, peptides were chemically constrained to be either he-
lical or ?-turn (17, 23), but broadly potent neutralizing anti-
bodies have not been produced from these immunogens. In
this regard, our data support an alternative strategy for elicit-
ing 2F5-like antibodies based on exposure of the 2F5 epitope
in the fusion intermediate.
Finally, the peptide-trapping studies suggested to us that the
mechanism of 2F5 neutralization does not involve fixing Env in
the native conformation but instead involves interfering with
Env-mediated entry at some later step, after initial receptor-
induced conformational changes. To investigate whether the
2F5 MAb neutralizes HIV-1 by preventing formation of the
six-helix bundle, we pretreated 500 ?l of CHO-Env-expressing
cells with 10.5 ?g of the 2F5 MAb before receptor activation (5
FIG. 3. Antibody binding to the fusion intermediate. 2F5 (A) and
D50 (B) MAbs were used to immunoprecipitate peptide-trapped Env
from gp41 expressed on cell surface with (?) or without (?) sCD4 in
the absence (?) or presence (?) of the C34 or N36 peptide. The C34
peptide binds the N heptad of gp41, and the N36 peptide binds the C
heptad of gp41, preventing formation of the six-helix bundle using
endogenous heptad repeats from gp41. Blots were probed with an
antibody to gp41. Results shown are representative of at least three
FIG. 4. Antibodies binding to the six-helix bundle after pretreatment with 2F5. (A) Increasing concentrations of the NC-1 MAb or anti-six-helix
bundle antibodies (?-b1, ?-b2) are able to immunoprecipitate gp41 equally well in the presence or absence of pretreatment with 2F5. (B) In-
creasing concentrations of 2F5 do not impair the ability of the NC-1 to precipitate gp41. Blots were probed with an antibody to gp41. Control is
an immunoblot of Env-expressing cell lysate. Results shown are representative of at least three independent experiments.
VOL. 78, 2004NOTES 2629
?l of sCD4) and immunoprecipitated gp41 with two sets of
anti-six-helix bundle antibodies (Fig. 4). The first antibody is
the NC-1 MAb (15), which was raised against a recombinant
gp41 (35); it binds a conformational epitope in the six-helix
bundle and is nonneutralizing (15). The second antibody set is
a pair of polyclonal anti-peptide sera (?-b1 and ?-b2) that
preferentially bind the six-helix bundle (6). It was previously
shown that sCD4 triggers formation of the fusion intermediate
and six-helix bundle with the HXB2 Env (11, 15). Presumably,
displacement of gp120 from gp41 by sCD4 is sufficient to allow
gp41 to fold into its most thermostable conformation, though
the HXB2 gp120 may be more easily released from gp41 than
some other HIV-1 strains. Surprisingly, the presence of high
concentrations of 2F5 had no effect on the ability of increasing
concentrations of the NC-1 (Fig. 4A, lanes 1 to 7) or the
purified sera against the six-helix bundle (Fig. 4A, lanes 9 to 12
and 15 to 18) to bind gp41. Similarly, when increasing amounts
of the 2F5 MAb were added to Env-expressing cells that were
subsequently incubated with NC-1 in the presence or absence
of sCD4, we detected no changes in the amount of gp41 im-
munoprecipitated by NC-1 (Fig. 4B, lanes 4 to 6). As shown in
negative control lines, we were able to selectively pull down
gp41 with the mouse NC-1 MAb (Fig. 4A, lane 7) or the rabbit
sera (Fig. 4A, lanes 13 and 19) rather than the human 2F5
MAb by using species-specific magnetic beads.
Together these results suggest that 2F5 does not strictly
prevent folding of gp41 into the six-helix bundle, nor does it
enhance six-helix bundle formation by prematurely triggering
the fusion-active state. These observations were unexpected
but can be explained in several ways. Although unlikely, we
cannot rule out the possibility that the NC-1 monoclonal and
our two sets of polyclonal antibodies recognize epitopes
present in an incomplete six-helix bundle that is not fully
formed. It is also possible that despite using high concentra-
tions of 2F5, only a small fraction of Env are bound by 2F5,
which would be enough to inhibit fusion but not enough to
reduce triggering of a majority of Envs. This scenario would
suggest that 2F5 neutralization is extremely efficient; 2F5 may
only need to bind a few Envs to neutralize virus. Nonetheless,
our data indicate that neutralization by 2F5 involves a novel
mechanism that apparently impairs a post-fusion-intermediate
step in the fusion process. Perhaps when membrane-bound
receptors activate Env, as happens during natural infection,
attachment of 2F5 to gp41 impedes close apposition of the
target and viral membranes and/or interaction of the ectodo-
main of gp41 with membranes. In this case, interference of
membrane merger by the antibody could impair complete for-
mation of the six-helix bundle (22) or interfere with post-six-
helix fusion events that involve bending and ordering of mul-
tiple fusion-active Envs to form a fusion pore. This hypothesis
is supported by recent reports of two other potent neutralizing
antibodies, 4E10 and Z13, which bind near the 2F5 epitope in
the pretransmembrane region (33, 41) and could conceivably
neutralize by a similar mechanism. 4E10, in contrast to 2F5,
binds a linear epitope in both native and triggered conforma-
tions (41; data not shown) and would therefore not be ex-
pected to directly prevent formation of the six-helix bundle.
In summary, we have used membrane-anchored Env and
antibodies in physiological conditions to show that the C-hep-
tad repeat region of gp41 undergoes conformational changes
after gp120 binds the receptor. We also demonstrated that 2F5
appears to bind both native and fusion-intermediate confor-
mations of gp41 and neutralizes virus at a relatively late step in
virus entry. These findings provide insights into the mechanism
of Env-mediated membrane fusion and suggest ways to inter-
fere with this process.
We thank Ira Berkower, Keith Peden, and Hana Golding (Center
for Biologics Evaluation and Research [CBER], Bethesda, Md.) for
critical reading of the manuscript, Nga Nguyen for peptide production
(CBER Facility for Biotechnology Resources, Bethesda, Md.), and Pat
Earl (NIH, Bethesda, Md.) for providing the D50 MAb.
We also acknowledge the support of the NIH Intramural AIDS
Targeted Antiviral Program.
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