JOURNAL OF VIROLOGY, Apr. 2010, p. 3147–3161
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 7
A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding
Stabilizes the Human Immunodeficiency Virus
Envelope Glycoprotein Trimer?
Shi-Hua Xiang,1Andre ´s Finzi,1Beatriz Pacheco,1Kevin Alexander,1Wen Yuan,1Carlo Rizzuto,1
Chih-Chin Huang,2Peter D. Kwong,2and Joseph Sodroski1,3*
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Pathology, Division of AIDS,
Harvard Medical School, Boston, Massachusetts 021151; Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922; and Department of
Immunology and Infectious Diseases, Harvard School of Public Health,
Boston, Massachusetts 021153
Received 10 December 2009/Accepted 5 January 2010
Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of
noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of
gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing
capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120
third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here
that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result
in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in
the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from
primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope
glycoproteins. Changes in the gp120 ?2, ?19, ?20, and ?21 strands, which evidence suggests are proximal to
the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element
composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the
unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering
the hydrophobic patch in the V3 tip available for chemokine receptor binding.
The entry of human immunodeficiency virus type 1 (HIV-1)
is mediated by the viral envelope glycoproteins (9, 79). The
HIV-1 envelope glycoproteins are synthesized as an ?850-
amino acid precursor, which trimerizes and is posttranslation-
ally modified by carbohydrates to create a 160-kDa glycopro-
tein (gp160). The gp160 envelope glycoprotein precursor is
proteolytically processed in the Golgi apparatus, resulting in a
gp120 exterior envelope glycoprotein and a gp41 transmem-
brane envelope glycoprotein (16, 17, 66, 76). In the mature
HIV-1 envelope glycoprotein trimer, the three gp120 subunits
are noncovalently bound to three membrane-anchored gp41
HIV-1 entry involves the binding of gp120 in a sequential
fashion to CD4 and one of the chemokine receptors, CCR5 or
CXCR4 (1, 8, 15, 18, 25, 36). CD4 binding triggers the forma-
tion of an activated intermediate that is competent for binding
to CCR5 or CXCR4 (29, 69, 73, 78). These chemokine recep-
tors are G protein-coupled, 7-transmembrane segment recep-
tors with relatively short N termini. The choice of chemokine
receptors is dictated primarily by the sequence of a gp120
region, the third variable (V3) loop, that exhibits variability
among HIV-1 strains and becomes exposed upon CD4 binding
(4, 8, 10, 33, 37, 38, 49, 59, 75). X-ray crystal structures of
CD4-bound HIV-1 gp120 have revealed that the gp120 “core”
consists of a gp41-interactive inner domain, a surface-exposed
and heavily glycosylated outer domain, and a conformationally
flexible bridging sheet (38, 43, 79). In the CD4-bound state, the
V3 loop projects 30 Å from the gp120 core, toward the che-
mokine receptor (38). The V3 loop in these structures consists
of three elements: (i) conserved antiparallel ? strands that
contain a disulfide bond at the base of the loop; (ii) a confor-
mationally flexible stem; and (iii) a conserved tip (37, 38).
During the virus entry process, the base of the gp120 V3 loop
and elements of the bridging sheet interact with the CCR5 N
terminus, which is acidic and contains sulfotyrosine residues
(12–14, 23, 24). Sulfotyrosine 14 of CCR5 is thought to insert
into a highly conserved pocket near the V3 base, driving fur-
ther conformational rearrangements that result in the rigidifi-
cation of the V3 stem (37). The conserved ?-turn at the tip of
the V3 loop, along with some residues in the V3 stem, is
believed to bind the “body” of CCR5, i.e., the extracellular
loops and membrane-spanning helices. CCR5 binding is
thought to induce further conformational changes in the
HIV-1 envelope glycoproteins, leading to the fusion of the viral
and target cell membranes by the gp41 transmembrane enve-
CCR5 binding involves two points of contact with the gp120
V3 loop: (i) the CCR5 N terminus with the V3 base and (ii) the
CCR5 body with the V3 tip and distal stem (12–14, 23, 24, 37,
38). The intervening V3 stem can tolerate greater conforma-
* Corresponding author. Mailing address: Dana-Farber Cancer In-
stitute, 44 Binney Street, CLS 1010, Boston, MA 02115. Phone: (617)
632-3371. Fax: (671) 632-4338. E-mail: email@example.com
?Published ahead of print on 20 January 2010.
tional and sequence variation, features that might decrease
HIV-1 susceptibility to host antibodies (30). Despite amino
acid variation, the length of the V3 loop is well conserved
among naturally occurring group M (major group) HIV-1
strains (30, 42). This conserved length may be important for
aligning the two CCR5-binding elements of the V3 loop. In
addition to allowing optimal CCR5 binding, the conserved V3
length and orientation may be important for CCR5 binding to
exert effects on the conformation of the HIV-1 envelope gly-
coproteins. We examine here the consequences of introducing
extra amino acid residues into the V3 stem. The residues were
introduced either into both strands of the V3 loop, attempting
to preserve the symmetry of the structure, or into one of the
strands, thereby kinking the loop. The effects of these changes
on assembly, stability, receptor binding, and the membrane-
fusing capacity of the HIV-1 envelope glycoproteins were as-
sessed. In addition to effects on chemokine receptor binding,
unexpected disruption of gp120-gp41 association was ob-
served. Further investigation revealed a conserved patch in the
tip of the V3 loop that is important for the association of gp120
with the trimeric envelope glycoprotein complex, as well as for
chemokine receptor binding. Apparently, the V3 loop and
adjacent gp120 structures contribute to the stability of the
trimer in the unliganded HIV-1 envelope glycoproteins. These
structures are known to undergo rearrangement upon CD4
binding, suggesting their involvement in receptor-induced
changes in the virus entry process.
MATERIALS AND METHODS
HIV-1 envelope glycoprotein and CCR5 mutants. Mutations were introduced
into the pSVIIIenv plasmid expressing the full-length envelope glycoproteins
from the ADA, YU2, 89.6, HXBc2, and MN27 HIV-1 strains (68), using a
QuikChange II XL site-directed mutagenesis protocol (Stratagene). The pres-
ence of the desired mutations was confirmed by DNA sequencing. The V3
insertion mutants are designated m1, m2, m3, etc. (Fig. 1). The gp120 substitu-
tion mutants are designated with the amino acid residue to the right of the
number substituted for the amino acid residue to the left of the number. All
residues are numbered according to those of the prototypic HXBc2 sequence, as
per current convention (41).
The predicted amino acid sequence of the HIV-1 ADA ?V3 mutant is:
296CTgagHC332, where the lower case gag represents a glycine-alanine-glycine
linker. The predicted amino acid sequence of the HIV-1 HXBc2 ?V3 and
?V3-20 mutants, respectively, are:
The CCR5-GG mutant contains a two-residue insertion in the N terminus of
human CCR5. The sequence of the CCR5-GG protein near the insertion is: . . .S
PIYDINYYTSEGGPCQKINV. . ., with the inserted residues underlined.
Syncytium formation assay. The ability of the HIV-1 envelope glycoprotein
variants to mediate cell-cell fusion was determined by the ?-complementation
assay (35). In this assay, the N-terminal (?) fragment of ?-galactosidase is
coexpressed with the HIV-1 envelope glycoproteins; the C-terminal (?) fragment
of ?-galactosidase is expressed in the receptor-bearing target cells. Upon fusion
of the envelope glycoprotein-expressing cells and the target cells, the ?-galacto-
sidase is reconstituted, and its activity can be measured. Briefly, 293T cells in
six-well plates were transfected with plasmids expressing the ?-galactosidase ?
fragment, the HIV-1 envelope glycoproteins and the HIV-1 Tat protein in a 4:4:1
weight ratio. Cf2Th-CD4/CCR5 cells (for the HIV-1 ADA, YU2, and 89.6
envelope glycoproteins) or Cf2Th-CD4/CXCR4 cells (for the HIV-1 89.6,
HXBc2, and MN27 envelope glycoproteins) in 100-mm dishes were transfected
with 3 ?g of a plasmid expressing the ?-galactosidase ? fragment. On the next
day, the Cf2Th target cells were lifted from the plates with 5 mM EDTA–
phosphate-buffered saline (EDTA-PBS) and reseeded at 105cells/well in 96-well
plates. One day later, the transfected 293T cells were lifted from the plates with
5 mM EDTA-PBS and diluted to 2 ? 105cells/ml with Dulbecco modified Eagle
medium (DMEM). Approximately 2 ? 104293T cells (in 100 ?l) expressing the
HIV-1 envelope glycoproteins were added in triplicate to each well of a 96-well
plate containing the target cells. The cocultivated cells were cultured at 37°C in
a CO2incubator for 4 h. The medium was then removed, and the cells were
washed once with PBS and lysed in 20 ?l of lysis buffer. The plate was covered
with Parafilm, wrapped in plastic film and place at ?70°C for 30 min. The
?-galactosidase activity was measured with a Galacto-Star ?-galactosidase re-
porter gene assay system for mammalian cells (Tropix, Bedford, MA), using a
Berthold Microplate Luminometer LB 96V (Promega).
The ability of the wild-type and mutant HIV-1 ADA envelope glycoproteins
expressed transiently in 293T cells to form syncytia with target Cf2Th-CD4 cells
expressing CD4 and either the wild-type CCR5 protein or the CCR5-GG mutant
was assessed by using the ?-complementation assay described above. The 293T
cells were transfected with 0.8 ?g of the envelope glycoprotein expressor plas-
mid, 0.8 ?g of the plasmid expressing the ?-fragment of ?-galactosidase, and 0.2
?g of the Tat-expressing plasmid. The Cf2Th-CD4 cells in 100-mm dishes were
transfected with 2.5 ?g of the plasmid expressing either wild-type CCR5 or
CCR5-GG and 2.5 ?g of the plasmid expressing the ?-fragment of ?-galactosi-
dase. On the next day, the transfected Cf2Th-CD4 cells were harvested with 5
mM EDTA-PBS and reseeded in a 96-well plate (2 ? 104cells/well). The similar
surface expression level of the wild-type CCR5 and the CCR5-GG proteins was
verified by fluorescence-activated cell sorting using phycoerythrin-conjugated
2D7 antibody (BD Pharmingen). The medium of the 293T cells was changed on
the day after transfection. The following day, the 293T cells were harvested with
5 mM EDTA-PBS, diluted to 2 ? 105cells/ml with DMEM, and then coculti-
vated with the Cf2Th-CD4 cells as described above. After 4 h, the cells were lysed
and ?-galactosidase activity measured, as described above.
In some cases, an additional assay was used to measure the syncytium-forming
ability of the HIV-1 envelope glycoprotein variants (Table 1). In this assay, cells
coexpressing the HIV-1 envelope glycoproteins and Tat were cocultivated with
TZM-bl cells. Cell-cell fusion was quantitated by measuring luciferase activity
(A. Finzi et al., unpublished data).
Infection by single-round luciferase-expressing HIV-1. Recombinant lucif-
erase-expressing HIV-1 viruses were produced by transfection of 293T cells with
the pCMV Gag-Pol packaging plasmid, the pHIV-luc vector and the pSVIIIenv
plasmids (50). At 3 days after transfection, the cell supernatants were harvested.
The amount of virus in the supernatants was assessed by measurement of reverse
transcriptase (RT) (57).
For infection, Cf2Th-CD4/CCR5 or Cf2Th-CD4/CXCR4 cells were plated at
a density of 6 ? 103cells/well in a 96-well plate. The following day, the cells were
incubated with 2,500 RT units of recombinant virus per well. Two days later, the
cells were lysed, and the luciferase activity measured in the Berthold microplate
luminometer LB 96V (Promega).
Immunoprecipitation of HIV-1 envelope glycoproteins. 293T cells were trans-
fected with pSVIIIenv plasmids expressing the HIV-1 envelope glycoproteins.
One day later, the cells were metabolically labeled for 16 h with35S-Protein
Labeling Mix (Perkin-Elmer). The cell lysates (containing gp160 and gp120) and
media (containing gp120) were used for immunoprecipitation. Briefly, 400 ?l of
clarified cell lysate or medium was incubated with 100 ?l of 10% protein A-
Sepharose beads (Amersham Biosciences) and 4 ?l of a mixture of a sera from
HIV-1-infected individuals. For some experiments, 1 ?g of a monoclonal anti-
body was used instead of the serum mixture. The mixtures were brought to a
volume of 1 ml with PBS and incubated on a shaking platform at room temper-
ature for 1 h. The immunoprecipitation of HIV-1 gp120-containing cell super-
natants by the G3-299 monoclonal antibody was carried out at 4°C for 2 h in the
presence of protease inhibitors (one tablet Complete protease inhibitor cocktail
[mini, EDTA-free] per 10-ml binding reaction; Roche Applied Science, Ger-
many). The pelleted beads were washed once with 0.5 M NaCl-PBS and twice
with PBS. The beads were then suspended in a 2? loading buffer, boiled, and
applied to a 10% sodium dodecyl sulfate-polyacrylamide gel.
CCR5 binding assay. To assess CCR5-binding ability, normalized amounts of
radiolabeled gp120 envelope glycoproteins from transfected 293T cell superna-
tants were incubated with sCD4 and Cf2Th-CCR5 cells. Briefly, Cf2Th-CCR5
cells were lifted from the plate by using 5 mM EDTA-PBS (pH 7.5). After a wash
with DMEM, the cells were resuspended in DMEM and added to 1.5-ml micro-
centrifuge tubes (3 ? 106cells/tube). The radiolabeled gp120-containing cell
supernatants (500 ?l) and 10 ?g of sCD4 were added to the tube, and the volume
was adjusted to 1 ml with DMEM. The tubes were rocked at room temperature
for 1 h. The cells were then washed once with PBS and lysed in 1 ml of 1? NP-40
buffer. The cell lysates were precipitated with a mixture of sera from HIV-1-
infected individuals and protein A-Sepharose beads (Amersham Biosciences) at
4°C for 2 h. The precipitated gp120 was analyzed by SDS-PAGE and autora-
3148 XIANG ET AL.J. VIROL.
HIV-1 V3 loop insertion mutants. To examine the effects of
changes in the length or orientation of the HIV-1 gp120 V3
loop, a panel of insertion mutants derived from the ADA
HIV-1 isolate was created (Fig. 1). The ADA virus is a pri-
mary, CCR5-using (R5) HIV-1 isolate (27). In one set of mu-
tants, one or two glycine residues were introduced into one
strand of the conformationally flexible stem of the V3 loop;
these asymmetric insertions would presumably kink the loop.
In a second set of mutants, identical insertions were placed
symmetrically into the N- and C-terminal strands of the V3
stem; the intent of these changes was to extend the length of
the V3 loop while minimizing any alterations in the orientation
of the tip.
The ability of the V3 insertion mutants to mediate the fusion
of envelope glycoprotein-expressing cells and cells bearing
CD4 and CCR5 was assessed. Plasmid DNAs expressing the
mutant envelope glycoproteins and the ? fragment of ?-galac-
tosidase were transfected into 293T cells. The 293T cells were
cocultivated with canine Cf2Th cells expressing human CD4,
human CCR5 and the ? fragment of ?-galactosidase. Success-
ful fusion of the Env-expressing cells with the Cf2Th-CD4/
CCR5 cells results in reconstitution of enzymatically active
?-galactosidase (35). In this cell-cell fusion assay, most of the
HIV-1 ADA V3 insertion mutants exhibited ?5% of the ac-
tivity observed for the wild-type ADA envelope glycoproteins
(Fig. 2A). Mutants 3, 4, 5, and 6 exhibited syncytium-forming
abilities between 5 and 60% of that seen for the wild-type
ADA envelope glycoproteins. These four mutants contain gly-
cine insertions in the C-terminal strand of the V3 stem.
To examine the generality of these results, V3 mutants 1 and
2 were created in the context of envelope glycoproteins derived
from two additional primary viruses, the R5 YU2 strain and
the dualtropic (R5X4) 89.6 strain (11, 47), and from two
CXCR4-using (X4) laboratory-adapted viruses, HXBc2 and
MN27 (26, 64) (Fig. 1). The syncytium-forming abilities of
these envelope glycoproteins were assessed as described above.
For the envelope glycoproteins from the HXBc2 and MN27
HIV-1 strains, the mutant 1 glycoproteins, which contain a
single glycine inserted into the N-terminal strand of the V3
stem, exhibited 80 to 85% of the syncytium-forming abilities of
the respective wild-type envelope glycoproteins (Fig. 2B). In
the context of the YU2 and 89.6 envelope glycoproteins, the
mutant 1 insertion more significantly reduced syncytium-form-
ing ability. Likewise, the HXBc2 and MN27 mutant 2 envelope
glycoproteins, which contain two glycine residues inserted into
the N-terminal strand of the V3 stem, exhibited ca. 50 and 70%
of the syncytium-forming ability of the respective wild-type
envelope glycoproteins. In contrast, the mutant 2 variants of
the YU2 and 89.6 envelope glycoproteins induced only very
low numbers of syncytia. The dualtropic 89.6 envelope glyco-
protein variants induced fusion with target cells expressing
CD4 and CCR5 or CD4 and CXCR4 equivalently. Thus, in-
sertions in the V3 stem generally reduced the ability of HIV-1
envelope glycoproteins to induce cell-cell fusion. The extent of
this reduction depended upon the number of residues inserted
FIG. 1. V3 loop insertion mutants. The V3 loop sequences of the wild-type (wt) and insertion mutant envelope glycoproteins from the indicated
HIV-1 strains are aligned. The numbering of the gp120 residues corresponds to that of the HXBc2 prototype, according to current convention (41).
The corresponding segments of the V3 loop structure in the CD4-bound state (38) are shown above the alignment. The inserted glycine residues
are shown in lower case.
VOL. 84, 2010HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY 3149
and upon the particular HIV-1 envelope glycoproteins altered,
with the laboratory-adapted HIV-1 envelope glycoproteins be-
ing functionally more tolerant of these changes than the pri-
mary HIV-1 envelope glycoproteins.
Infectivity of HIV-1 with V3 loop insertions. The ability of
the V3 insertion mutants to support HIV-1 infection was as-
sessed in a single-round Env complementation assay (31). Re-
gardless of the HIV-1 strain from which the envelope glyco-
proteins were derived, all of the V3 mutants were markedly
defective in mediating HIV-1 entry (Fig. 2C and D). Complete
defectiveness was also observed for an ADA envelope glyco-
protein mutant with an alanine substitution in place of the
glycine insert in mutant 1 (data not shown).
Expression, processing, and subunit association of the
HIV-1 Env mutants. To investigate the basis for the reduced
activities of the V3 mutants in syncytium-forming ability and
virus replication, cells transiently expressing the wild-type
and mutant envelope glycoproteins were radiolabeled. Cell
lysates and supernatants were precipitated by a polyclonal
mixture of sera from HIV-1-infected individuals. In the cell
lysates, the wild-type gp160 envelope glycoprotein precursor
and the mature gp120 envelope glycoprotein were evident
(Fig. 3A and B). For the primary HIV-1 ADA, YU2, and
89.6 envelope glycoproteins, although the levels of the V3
mutant gp160 envelope glycoproteins were generally similar
to those of the wild-type counterparts, the levels of cell-
associated gp120 glycoproteins were relatively reduced (Fig.
3A and B). For these HIV-1 envelope glycoproteins, the
amounts of gp120 shed into the medium were increased for
the V3 mutants compared to the wild-type glycoproteins.
These results suggest that the V3 loop insertions decrease
the association of the gp120 and gp41 subunits in the unli-
ganded envelope glycoprotein complex. The amount of cell-
associated gp120 glycoprotein was lower for the wild-type
HXBc2 and MN27 envelope glycoproteins than for the pri-
mary HIV-1 envelope glycoproteins, under these labeling
conditions (Fig. 3B). Slight decreases in the amount of cell-
associated gp120 were observed for the HXBc2 m1 and m2
mutants, relative to the wild-type HXBc2 envelope glyco-
proteins; however, no differences between the phenotypes of
the MN27 wild-type and mutant envelope glycoproteins with
respect to gp160 precursor processing or gp120-gp41 asso-
ciation were evident. Thus, the V3 loop insertions decrease
gp120-gp41 association of primary HIV-1 envelope glyco-
FIG. 2. Syncytium-forming activity and ability of V3 insertion mutants to support HIV-1 infection. (A) 293T cells expressing the wild-type (wt)
or the indicated HIV-1 ADA envelope glycoprotein mutants were cocultivated with Cf2Th-CD4/CCR5 cells and syncytium formation measured
as described in Materials and Methods. The negative control cells were transfected with a plasmid expressing the HIV-1 HXBc2 envelope
glycoproteins. The syncytium-forming activity of the mutants is reported relative to that seen for the wild-type HIV-1 ADA envelope glycoproteins.
Means and standard deviations derived from four replicate assays are shown. (B) The relative syncytium-forming activities of the mutant 1 and 2
envelope glycoproteins are shown for each of the indicated HIV-1 strains. Cf2Th-CD4/CXCR4 target cells were used for the HXBc2, MN27 and
89.6 envelope glycoproteins, and Cf2Th-CD4/CCR5 target cells were used for the YU2 and 89.6 envelope glycoproteins. The experiments were
conducted as described in panel A. The means and standard deviations derived from four replicate assays are shown. (C and D) The wild-type (wt)
or mutant (m1, m2, etc.) envelope glycoproteins from the indicated HIV-1 strain were assessed for the ability to support the infection of
recombinant HIV-1 expressing luciferase, using Cf2Th-CD4/CCR5 target cells (for the ADA, YU2, and 89.6 HIV-1 envelope glycoproteins) or
Cf2Th-CD4/CXCR4 target cells (for the HXBc2 and 89.6 HIV-1 envelope glycoproteins). The infectivity of the mutants relative to that seen for
the respective wild-type envelope glycoproteins is shown. The experiments were repeated with comparable results.
3150 XIANG ET AL.J. VIROL.
proteins; this phenotype is less evident for envelope glyco-
proteins derived from laboratory-adapted HIV-1 isolates.
Decreased stability of the envelope glycoprotein complex
explains the lower function of the V3 loop mutants in mediat-
ing virus entry compared to cell-cell fusion. Because a greater
time interval elapses between envelope glycoprotein synthesis
and engagement of the target cell in the virus entry assay,
decreases in the functional stability of the HIV-1 envelope
glycoproteins are more apparent than in the syncytium forma-
tion assay (84; Finzi et al., unpublished).
Interaction of mutant envelope glycoproteins with ligands.
To assess the effect of the V3 loop insertions on gp120 con-
formation, the recognition of the gp120 glycoprotein by con-
formation-dependent ligands was assessed. Radiolabeled
gp120 glycoproteins from transfected cell supernatants were
precipitated by a mixture of sera from HIV-1-infected individ-
uals, which recognizes gp120 independently of its conforma-
tional integrity, or by specific gp120-directed ligands whose
recognition depends upon gp120 conformation. The ligands
include: CD4-Ig, in which the amino-terminal two domains of
CD4 are fused to an immunoglobulin Fc; 17b and 412d, two
antibodies that recognize CD4-induced epitopes near the che-
mokine receptor-binding surface of gp120 (58, 72); and 39F,
which recognizes a conformation-dependent V3 epitope (45).
All of the HIV-1 ADA gp120 mutants tested bound CD4
efficiently, as evidenced by the precipitation of these glycopro-
teins by CD4-Ig (Fig. 4A). The 17b and 412d antibodies pre-
cipitated all of the gp120 mutants, although the recognition of
some of the mutants by the 17b antibody was less efficient than
that of the wild-type gp120 glycoprotein. This result is consis-
tent with the proximity of the 17b epitope to the base of the V3
loop (43, 58). The 39F anti-V3 antibody precipitated most of
the mutants equivalently to the wild-type gp120, with three
exceptions. Mutants 4, 6, and 9 were precipitated less effi-
ciently than the wild-type gp120 glycoprotein by the 39F anti-
body. All three mutants have a two-residue insertion in the
carboxy-terminal half of the V3 stem, suggesting that changes
in this V3 region can disrupt the 39F epitope. These results
suggest that the gp120 V3 insertion mutants are not globally
misfolded but exhibit specific alterations near or within the V3
The ability of the gp120 mutants to bind CCR5 was exam-
ined (78). Radiolabeled gp120 glycoproteins from transfected
cell supernatants were incubated in the presence of soluble
CD4 with Cf2Th cells expressing CCR5. After a washing step,
the amount of gp120 bound to the cells was determined. Figure
4B shows that all of the V3 insertions decreased the efficiency
of CCR5 binding. This decrease was readily apparent for the
gp120 glycoproteins from the R5 HIV-1 strains, ADA and
YU2. The wild-type gp120 glycoprotein from the R5X4 (dual-
tropic) 89.6 HIV-1 strain exhibits a lower affinity for CCR5
than those of R5 gp120 glycoproteins (2, 3). Therefore, higher
concentrations of the 89.6 gp120 are required to demonstrate
CCR5 binding. At these higher concentrations, mutants 1 and
2 from the 89.6 strain bound CCR5 less efficiently than the
wild-type 89.6 gp120 glycoprotein (Fig. 4B). Thus, insertions
into the V3 stem of the HIV-1 gp120 glycoprotein result in
significant decreases in the efficiency of CCR5 binding.
Partial compensation of the syncytium-forming ability of V3
insertion mutants by a CCR5 protein with an extended amino
terminus. Current models of HIV-1 gp120-CCR5 interaction
suggest that the CCR5 N terminus binds near the V3 base and
the body of CCR5 binds the V3 tip (12–14, 23, 24, 37, 38).
Thus, the gp120 mutants with insertions in the V3 stem might
utilize CCR5 less efficiently because of poor alignment of these
two binding contacts. In this case, the mutants might be more
effective if the CCR5 N terminus were extended further from
the body of the chemokine receptor. To test this, two glycine
FIG. 3. Processing and subunit association of HIV-1 envelope glycoprotein variants. (A to D) 293T cells expressing the wild-type (wt) or mutant
envelope glycoproteins from the indicated HIV-1 strains were radiolabeled. The cells were pelleted and lysed. The radiolabeled cell lysates and
media were precipitated by a mixture of sera from HIV-1-infected individuals. The envelope glycoproteins precipitated from the cell lysates and
media were analyzed by SDS-PAGE. The gp160 and gp120 envelope glycoproteins are indicated.
VOL. 84, 2010HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY 3151
residues were inserted into the CCR5 sequence, between the N
terminus and the initial cysteine residue. This mutant, CCR5-
GG, and wild-type CCR5 were expressed in Cf2Th-CD4 cells,
which were assessed for the ability to form syncytia with cells
expressing the wild-type or mutant ADA envelope glycopro-
teins. The syncytium-forming ability of some of the V3 mutant
envelope glycoproteins, relative to that of the wild-type ADA
envelope glycoproteins, was greater with target cells expressing
the CCR5-GG mutant than with target cells expressing wild-
type CCR5 (Fig. 5). This phenotype was particularly evident
for envelope glycoprotein mutants (mutants 7, 8, and 9) with
symmetrical substitutions in the V3 loop and was also seen for
mutant 1 with a single glycine insertion. The defective entry of
viruses with the mutant envelope glycoproteins was not com-
pensated by the expression of the CCR5-GG mutant on the
target cell (data not shown). These results are consistent with
a model suggesting that one of the consequences of the V3
insertions is a disruption of the optimal spacing between gp120
regions important for binding the CCR5 N terminus and body.
A conserved V3 element that is important for gp120-gp41
association. The effect of V3 loop insertions on gp120-gp41
association could result from an incompatibility of a kinked or
extended loop with the proper packing of the gp120 subunits in
the trimeric spike. Alternatively, specific structures in the V3
loop could positively contribute to subunit association. Al-
though the V3 region is not absolutely essential for gp120-gp41
association in the laboratory-adapted HXBc2 envelope glyco-
proteins (see Fig. 3C), mild increases in the amount of gp120
spontaneously shed into the medium have been observed for
V3 loop-deleted envelope glycoproteins compared to the wild-
type HXBc2 envelope glycoproteins (81). To examine the role
of the V3 loop in maintaining the integrity of the trimeric
envelope glycoprotein complex in a primary HIV-1 isolate, the
V3 loop of the ADA gp120 glycoprotein was deleted in pre-
cisely the same manner as that used previously for the HXBc2
envelope glycoproteins from a laboratory-adapted HIV-1. The
V3 deletion resulted in a decrease in gp120-gp41 association
comparable to that which resulted from an insertion into the
V3 region (see Fig. 3D). Thus, the absence of the V3 loop can
weaken gp120-gp41 interactions in the envelope glycoprotein
trimer of a primary HIV-1 isolate.
The result described above raised the possibility that a V3
loop element contributes in a positive way to subunit associa-
tion. Our observation that V3 insertions decreased the stability
of the envelope glycoprotein trimers from multiple primary
HIV-1 strains suggested that such a V3 element might be
conserved in different HIV-1 isolates. Thus, we shifted our
attention away from the more variable V3 stem to the rela-
tively conserved V3 tip. Although the structure of the V3 loop
in the unliganded HIV-1 envelope glycoproteins is unknown,
in the CD4-bound gp120 (38), a ?-hairpin at the V3 tip juxta-
poses residues 307, 309, and 317 (Fig. 6, column at far right).
Some variability is tolerated in these residues in HIV-1 strains;
however, the hydrophobic character of these residues is almost
always maintained (Fig. 7A). We hypothesized that this hydro-
phobic patch in the V3 tip serves to strengthen gp120-gp41
association in the unliganded HIV-1 envelope glycoprotein
trimer. Amino acid residues of different degrees of hydropho-
bicity were introduced into residues 307, 309, and 317 of the
HIV-1 YU2 envelope glycoproteins. Although hydrophobic
substitutions did not significantly disrupt gp120-gp41 associa-
tion, substitutions of alanine or hydrophilic residues resulted in
severe decreases in subunit association (Fig. 7B to D). Thus,
the hydrophobic patch in the V3 tip contributes to subunit
association in the unliganded HIV-1 envelope glycoprotein
Contribution of the V3 hydrophobic patch to HIV-1 enve-
lope glycoprotein function. The V3 tip is known to contribute
to CCR5 binding (8, 9, 12, 14, 34, 58). The effect of changes in
the hydrophobic V3 patch on CCR5 binding and syncytium
formation were examined. Decreases in CCR5-binding affinity
resulted from hydrophilic substitutions in residues 307, 309,
and 317 (Fig. 4C and Table 1). These decreases were accom-
panied by defects in the ability to mediate the formation of
syncytia (Table 1). The effects of changes in the hydrophobic
V3 patch were in some cases more disruptive of gp120-gp41
association than of CCR5 binding and syncytium formation; in
FIG. 4. Binding of ligands to HIV-1 envelope glycoprotein variants.
(A) 293T cells expressing the wild-type (wt) or mutant (m1 to m9)
HIV-1 ADA envelope glycoproteins were radiolabeled. The cell su-
pernatants were precipitated by a mixture of sera from HIV-1-infected
individuals (PS), by CD4-Ig, or by the indicated monoclonal antibody.
The gp120 glycoproteins precipitated from the media were analyzed by
SDS-PAGE and autoradiography. (B and C) Radiolabeled wild-type
(wt) or mutant gp120 envelope glycoproteins from the indicated HIV-1
strains were incubated at room temperature for 1 h with Cf2Th-CCR5
cells in the presence of sCD4. The cells were washed and lysed, and the
bound gp120 was precipitated by a mixture of sera from HIV-1-in-
fected individuals. The bound gp120 glycoprotein was analyzed by
SDS-PAGE and autoradiography.
3152XIANG ET AL.J. VIROL.
general, however, the phenotypic effects of the V3 changes on
all of these properties correlated.
Involvement of V3-proximal gp120 regions in association
with gp41. Previous studies have suggested that, in the unli-
ganded gp120 glycoprotein, the V3 loop may interact with
other gp120 regions, particularly the ?19, ?20, and ?21 strands
(the fourth conserved [C4] region) and the V1/V2 stem-loop
structure (51–53, 67, 74, 80). Several residues conserved among
the gp120 glycoproteins of different HIV-1 strains were iden-
tified in these regions. The effect of alteration of these residues
in the HIV-1YU2envelope glycoproteins on the association of
gp120 with gp41 was examined (Table 1). Some changes in the
?19, ?20, and ?21 strands resulted in decreased subunit asso-
ciation (Table 1 and Fig. 7D). In addition, changes in leucine
120 and valine 122 in the ?2 strand of the conserved V1/V2
stem also reduced gp120-gp41 association. Thus, changes in
several HIV-1 gp120 regions that may be proximal to the V3
loop in the unliganded state resulted in decreases in the asso-
ciation of gp120 with gp41.
Previous studies have examined the effect of the HIV-1
gp120 changes on gp120-gp41 association (32, 63, 71, 84; Finzi
et al., unpublished). The results of these studies, as well as the
results generated in the present study, are summarized in Fig.
6. The N and C termini and inner domain of gp120, particularly
the inner domain ?-sandwich, are the major contributors to the
noncovalent interaction with gp41 (43, 55, 63, 84; Finzi et al.,
unpublished). Our results indicate that other gp120 regions
(the V3 loop, ?2, ?17, ?19, ?20, and ?21) can also influence
the stability of the association of gp120 and gp41. A compar-
ison of the different crystallized gp120 structures indicates that
these regions can change conformation in response to ligand
binding or alterations introduced into gp120 to promote crys-
tallization. Although the structure of the unliganded HIV-1
gp120 glycoprotein is unknown, in light of this conformational
flexibility, these gp120 elements are potentially in proximity in
the unliganded envelope glycoprotein trimer.
Effects of gp120 changes on a discontinuous V3-C4 epitope.
The G3-299 monoclonal antibody has been previously
shown to recognize a discontinuous HIV-1 gp120 epitope
that is apparently composed of elements from the V3 loop
and the fourth conserved (C4) region (51, 52). The G3-299
antibody neutralizes laboratory-adapted HIV-1 strains (60,
FIG. 5. Syncytium-forming ability of V3 insertion mutants with target cells expressing a CCR5 mutant. (A to C) 293T cells expressing the
wild-type (wt) or V3 mutant envelope glycoproteins from the indicated HIV-1 strain were cocultivated with Cf2Th cells expressing CD4 and either
wild-type (wt) CCR5 (white) or the CCR5-GG mutant (gray). CCR5-GG has two glycines inserted after residue 18, thus extending the N terminus.
Syncytium-forming ability was measured as described in Materials and Methods. In panels A and B, the syncytium-forming abilities of the envelope
glycoproteins are all normalized to that of the wild-type HIV-1 envelope glycoproteins on target cells expressing CD4 and wild-type CCR5. In panel
C, the syncytium-forming abilities of the mutant envelope glycoproteins are normalized to that of the wild-type envelope glycoproteins on target
cells expressing CD4 and the corresponding CCR5 variant. The means and standard deviations derived from three experiments are shown. The
delta V3 envelope glycoprotein is missing the gp120 V3 loop and is a fusion-defective negative control (81). 293T cells transfected with the empty
pcDNA vector serve as an additional negative control.
VOL. 84, 2010 HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY3153
70), indicating that this epitope is both intact and exposed
on the unliganded envelope glycoprotein trimers of at least
some HIV-1 variants. The effect of gp120 changes on the
integrity of this epitope was examined by precipitation of a
large panel of wild-type and mutant gp120 envelope glyco-
proteins by the G3-299 antibody. Of note, changes in the V3
loop (residues 307, 309, and 317) or in the ?21 strand
(residues 434 and 435) significantly decreased HIV-1 gp120
recognition by the G3-299 antibody (Tables 1 and 2). In the
CD4-bound state, the only gp120 conformation for which a
V3 loop structure is available (37, 38), these two elements
are separated (Fig. 8). This is consistent with the observa-
tion that CD4 binding decreases the recognition of HIV-1
gp120 by the G3-299 antibody (52).
The contribution of V3 loop and ?21 residues to the G3-299
epitope suggests that, in the unliganded HIV-1 gp120 glyco-
protein, these gp120 elements are proximate and form a dis-
continuous structural element. To gain additional insight into
this possibility, we examined the structure of HIV-1 gp120
bound to F105, a CD4-binding site (CD4BS) antibody that
recognizes a gp120 conformation distinct from that seen by
CD4 (6, 83). F105 and G3-299 can simultaneously bind gp120
(6), indicating that G3-299 can recognize the F105-bound con-
formation. With one exception, serine 375 (discussed below),
the gp120 residues implicated in G3-299 recognition are dis-
tinct from those that contact F105 (Fig. 8). In the F105-gp120
crystal, the V3 loop is intact but disordered (6). Nonetheless, in
this conformation, the V3 loop projects toward the ?21 strand,
potentially interacting with the ?21 residues implicated in G3-
299 binding (Fig. 8).
Substitution of a tryptophan residue for serine 375 resulted
in a decrease in the recognition of gp120 by the G3-299 anti-
FIG. 6. Changes in HIV-1 gp120 resulting in decreased association with gp41. A ribbon diagram of the unliganded simian immunodeficiency
virus (SIV) gp120 core structure (5) is shown in the left column. In the three columns on the right, the HIV-1 gp120 glycoprotein is shown in the
different conformations observed in available crystal structures, complexed with the Fab fragments of the b12 or F105 neutralizing antibodies (6,
87), or with two-domain CD4 (38). The gp120 structures are viewed from the perspective seen by the Fab or CD4 proteins; the outer domains of
the gp120 cores are aligned. The trimeric axis of the envelope glycoprotein complex is located on the left side of each structure, in approximately
the vertical orientation (44, 48). The gp120 core in the b12-bound structure was modified to predispose the protein to assume the CD4-bound
conformation (87). In the top row, the HIV-1 domains are colored as follows: outer domain (yellow), inner domain (red) and bridging sheet
components (blue for the ?20-?21 loop and green for the ?2-?3 V1/V2 stem). In the cases where the V3 loop structure was not determined, the
position of the V3 base is indicated. In the CD4-bound structure, the elements of the V3 loop are labeled. The gp120 beta strands (defined in
the CD4-bound structure ) relevant to the present study are also labeled. In the bottom row, the gp120 residues are colored according to the
gp120-gp41 association index (red, association index ? 0.5; green, association index ? 0.5) observed upon mutagenesis of the HIV-1YU2 and
HXBc2 gp120 glycoproteins (32, 63, 71, 84; the present study; Finzi et al., unpublished). In the CD4-bound gp120 structure, the three hydrophobic
residues in the tip of the V3 loop that were implicated in gp120-gp41 association are labeled.
3154XIANG ET AL.J. VIROL.
body (see Table 1 footnotes). Because serine 375 contacts the
F105 antibody, which does not compete with G3-299 for gp120
binding (6), the effects of the S375W change on G3-299 bind-
ing are likely indirect. Indeed, it has been shown that the
S375W mutant favors the CD4-bound conformation (83),
which is recognized less efficiently by the G3-299 antibody (52).
The V3 loop and adjacent gp120 elements in the unliganded
HIV-1 envelope glycoprotein trimer. The X-ray crystal structure
FIG. 7. The hydrophobic V3 patch. (A) The V3 variable loops of the primate immunodeficiency viruses are aligned. The degree of variation in
residues 307, 309, and 317 in the HIV-1 gp120 V3 tip is shown (42). The percentage of sequences in each virus group that contain the indicated residue
is shown as a superscript to the right of the single-letter amino acid designation. Amino acid variants that are found in less than 1% of the sequences
surveyed are not listed. (B to D) 293T cells expressing the wild-type (wt) or mutant HIV-1 YU2 envelope glycoproteins were radiolabeled. The cells were
pelleted and lysed. The radiolabeled cell lysates and media were precipitated by a mixture of sera from HIV-1-infected individuals. The envelope
glycoproteins precipitated from the cell lysates and medium were analyzed by SDS-PAGE. The gp160 and gp120 envelope glycoproteins are indicated.
VOL. 84, 2010 HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY3155
of the CD4-bound HIV-1 gp120 envelope glycoprotein with an
intact V3 loop (38) has been fitted to tomograms derived from
cryo-electron microscopy studies of HIV-1 virion spikes (48). In
the CD4-bound state, the exposed V3 loop projects obliquely
from the outer domain toward both the trimer axis and the target
cell (Fig. 6). Although the detailed structure of unliganded HIV-1
gp120 with an intact V3 loop is unknown, low-resolution electron
cryotomograms of the unliganded HIV-1 virion spike are avail-
be accommodated within the electron density of the unliganded
HIV-1 envelope glycoprotein spike (48). Thus, the structure ob-
served in the crystallized SIV gp120 core is distinct from that in
or artifactual differences between the unliganded SIV and HIV-1
gp120 glycoproteins. Both the CD4-bound and the b12 antibody-
bound HIV-1 gp120 core crystal structures can be readily fitted to
the electron cryotomograms of the unliganded HIV-1 envelope
glycoprotein spike (6, 48). The position and orientation of the V3
base in these models and the deduced proximity of the V3 loop to
the ?21 strand in the unliganded HIV-1 envelope glycoproteins
(see above) suggests that the V3 loops likely project toward the
trimer axis (Fig. 9 and data not shown). Thus, in the unliganded
HIV-1 envelope glycoproteins, the three V3 loops are potentially
poised for interactions with gp41 and/or the other gp120 elements
implicated in stabilizing the association with gp41.
Here we show that the insertion of amino acid residues into
the stem of the V3 variable loop of the gp120 envelope glyco-
TABLE 1. Phenotypes of HIV-1YU2mutants
aThe processing and association indices were determined by precipitation of radiolabeled cell lysates and supernatants with mixtures of sera from HIV-1-infected
individuals. The association index is a measure of the ability of the mutant gp120 molecule to remain associated with the envelope glycoprotein complex on the
expressing cell, relative to that of the wild-type envelope glycoproteins. The association index is calculated as follows: association index ? (?mutant gp120?cell?
?wild-type gp120?supernatant)/(?mutant gp120?supernatant? ?wild-type gp120?cell). The processing index is a measure of the conversion of the mutant gp160 envelope
glycoprotein precursor to mature gp120, relative to that of the wild-type envelope glycoproteins. The processing index was calculated by the formula: processing index ?
(?total gp120?mutant? ?gp160?wild type)/(?gp160?mutant? ?total gp120?wild type). Additional HIV-1 YU2 gp120 mutants that exhibited association indices of ?0.20 were
K121D, R298G, E381R, K421A, and P437A (data not shown). ND, not determined.
bRadiolabeled wild-type and mutant gp120 glycoproteins in the supernatants of envelope-expressing 293T cells were incubated with various amounts of sCD4-Ig for
2 h at 37°C in the presence of 70 ?l of 10% protein A-Sepharose (American Biosciences). At a near-saturating concentration of sCD4-Ig for the wild-type gp120
glycoprotein, the relative ratio of the mutant gp120 glycoprotein precipitated is reported. ND, not determined.
cThe CCR5-binding ability of the HIV-1 gp120 envelope glycoprotein variants was determined as described in Materials and Methods. The amount of bound mutant
gp120 glycoprotein was compared to the amount of bound wild-type gp120 glycoprotein. The relative CCR5-binding ability is reported as follows: ????, 75 to 100%
of the wild-type gp120 level; ???, 50 to 74% of the wild-type level; ??, 25 to 49% of the wild-type level; ?, 5 to 24% of the wild-type level; and –, ?5% of the wild-type
level. ND, not determined.
dTo assess cell-to-cell fusion, 3 ? 105293T cells were cotransfected by the calcium phosphate method with an HIV-1 Tat-expressing plasmid, pLTR-Tat, and the
pSVIIIenv plasmid expressing the HIV-1YU2envelope glycoproteins. At 2 days after transfection, 3 ? 104293T cells were added to TZM-bl target cells that were seeded
at a density of 3 ? 104cells/well in 96-well luminometer-compatible tissue culture plates (Dynex) 24 h before the assay. Cells were coincubated for 6 h at 37°C, after
which they were lysed by the addition of 30 ?l of passive lysis buffer (Promega) and three freeze-thaw cycles. The luciferase activity in each well was measured as
described above. The reported value represents the ratio of the luciferase activity observed for the mutant envelope glycoproteins relative to that of the wild-type
envelope glycoproteins. ND, not determined.
eRadiolabeled wild-type and mutant gp120 glycoproteins in the supernatants of 293T cells expressing the HIV-1 YU2 envelope glycoproteins were precipitated
by the G3-299 antibody for 2 h at 4°C in the presence of Complete protease inhibitor cocktail (Roche Applied Science). Precipitates were analyzed as described
in Materials and Methods. At a near-saturating concentration of G3-299 antibody for the wild-type gp120 glycoprotein, the relative ratio of the mutant gp120
glycoprotein precipitated is reported. Relative values for G3-299 recognition for mutant HIV-1 YU2 gp120 glycoproteins not shown in the table were as follows:
m1, 1.07; m2, 0.77; H66A, 1.91; W69L, 2.17; L111A, 0.59; S375W, 0.26; H66A/S375W, 0.55; W69L/S375W, 0.73; and L111A/S375W, 0.60. ND, not determined.
3156XIANG ET AL.J. VIROL.
protein from primary HIV-1 isolates resulted in decreased sub-
unit association in the envelope glycoprotein trimer and de-
creased chemokine receptor binding. Similar phenotypes were
observed for alteration of a conserved hydrophobic patch in the
V3 tip. The displacement of the V3 hydrophobic patch by the V3
stem insertions represents a unifying explanation of the pheno-
types, but more direct contributions of the V3 stem to subunit
association and chemokine receptor binding are formally possi-
ble. The results support functional roles for the V3 loop in both
the unliganded state (i.e., maintaining subunit association) and
the CD4-bound state (i.e., chemokine receptor binding).
Detailed structures of the assembled HIV-1 envelope glyco-
proteins are not yet available to explain precisely how V3 loop
alterations lead to disruption of the gp120-gp41 interaction in
the unliganded envelope glycoprotein trimer. Not even the
structure of an unliganded gp120 monomer with an intact
gp41-interactive inner domain or V3 loop is available for guid-
ance. Nonetheless, X-ray crystal structures of gp120 in various
conformations, in some cases induced by the binding of neu-
tralizing antibodies, have been fitted into low-resolution cryo-
electron tomograms of the unliganded HIV-1 virion spike (6,
48, 55). These models are consistent with the expected place-
ment of conserved and variable gp120 surfaces, glycosylation
sites, epitopes for neutralizing and non-neutralizing antibodies
and receptor-binding regions (44). In these models, the V3
loop projects from the gp120 outer domain toward the trimer
axis. Fixation of the assembled HIV-1 envelope glycoproteins,
but not the gp120 monomer, by treatment with chemical cross-
linkers has been shown to decrease specifically the binding of
antibodies directed against the V3 loop and the gp41-interac-
tive face of gp120 (85). These observations are consistent with
the close packing of the V3 loops of the gp120 subunits near
the trimeric axis of the unliganded HIV-1 envelope glyco-
protein complex. This arrangement may help in sequestering
the CCR5-binding elements of the V3 loop away from poten-
tial recognition by neutralizing antibodies.
The requirement to avoid the binding of potentially neutral-
izing antibodies constrains the structure of the unliganded
envelope glycoproteins from primary HIV-1 isolates. Such con-
straints are removed by extensive passage of HIV-1 in tissue-
cultured cell lines (56, 61, 68, 77). Short of destabilizing gp120-
gp41 association, V3 variation may modulate quaternary
subunit interactions that determine sensitivity to neutralizing
antibodies. Indeed, in one case, a dualtropic HIV-1 retained
some function after truncation of the V3 loop but was more
sensitive to neutralization by antibodies (46). Likewise,
changes in V3 can determine differences in sensitivity to neu-
tralizing antibodies, even those directed against conserved
gp120 epitopes, between primary and laboratory-adapted
HIV-1 isolates (67, 86). V3 changes have also been shown to
account for the increased neutralization resistance of simian-
human immunodeficiency viruses passaged in monkeys (7, 21).
Thus, alterations in the gp120 V3 loop can modulate sensitivity
to neutralization by antibodies directed against multiple enve-
lope glycoprotein determinants.
The syncytium-inducing function of the envelope glyco-
proteins was disrupted to a greater extent by V3 loop insertions
for primary HIV-1 isolates compared to laboratory-adapted
viruses. Deletion of the V3 loop exerted an effect on gp120-
gp41 association that was greater for the primary HIV-1 ADA
envelope glycoproteins than for the laboratory-adapted
HXBc2 envelope glycoproteins. Several studies have suggested
that the V3 loops of laboratory-adapted HIV-1 envelope gly-
coproteins are more exposed than the V3 loops of primary
virus envelope glycoproteins; in some cases, these differences
were apparent even on the monomeric gp120 glycoproteins (4,
20, 28, 46, 49, 65, 85). The decreased accessibility of the primary
with other gp120 elements that ultimately modulate quaternary
interactions on the trimer. Based on the phenotypic effects of
gp120 amino acid changes on gp120-gp41 association, candidates
for these gp120 elements include the V1/V2 stem (?2), ?20-?21,
and ?17-?19. Perhaps, in the unliganded trimer, structures
formed by the interaction of V3 with these elements help to
create a stable binding interface with gp41, either by direct con-
inclusion of these regions in the discontinuous epitope for the
G3-299 monoclonal antibody, which recognizes the unliganded
HIV-1 gp120 monomer better than the CD4-bound gp120 (51,
52). Changes in gp120 residues 307, 309, and 317 in the hydro-
TABLE 2. Ligand binding by HIV-1 gp120 variants
aThe wt? protein and the wt? mutants in the table are derived from the
HIV-1 YU2 gp120 glycoprotein. Relative to gp120, the wt? protein and the
mutant wt? proteins have a ?82 N-terminal deletion and a ?128-194 deletion of
the V1/V2 variable loops (58).
bThe relative values for CD4 and CCR5 binding were taken from Rizzuto et
al. (58). ND, not determined.
cRadiolabeled wild-type and mutant gp120 glycoproteins in the supernatants
of 293T cells expressing the HIV-1 YU2 envelope glycoproteins were precipi-
tated by the G3-299 antibody for 2 h at 4°C in the presence of Complete protease
inhibitor cocktail (Roche Applied Science). Precipitates were analyzed as de-
scribed in Materials and Methods. At a near-saturating concentration of G3-299
antibody for the wild-type gp120 glycoprotein, the relative ratio of the mutant
gp120 glycoprotein precipitated is reported.
VOL. 84, 2010HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY3157
phobic patch in the V3 tip and in methionine 434 in ?21 specif-
ically disrupted the G3-299 epitope. A poorly replicating simian
immunodeficiency virus altered in a single ?21 residue (equiva-
lent to methionine 434 in HIV-1) reverted by changing the equiv-
alent of V3 residue 307, further suggesting that the V3 hydropho-
bic patch may be proximal to ?21 in the unliganded envelope
glycoproteins (53). Changes in the V3 hydrophobic patch and a
V1/V2 loop segment near the V1/V2 stem have recently been
shown to disrupt the HIV-1 trimer-specific epitopes recognized
by the broadly neutralizing PG9 and PG16 antibodies (74). This
observation supports the proximity of the V3 tip and the V1/V2
to the trimer axis to be influenced by quaternary interactions
among the subunits of the unliganded HIV-1 envelope glycopro-
The multiple intramolecular contacts required to maintain
trimer integrity may impose limitations on the tolerance of
primary HIV-1 envelope glycoproteins to V3 loop insertions
and deletions. Despite variation in particular amino acid resi-
dues, the length of the gp120 V3 loop is very well conserved
among primary HIV-1 strains, with rare exceptions (42). For
example, in the envelope glycoproteins of some group M
HIV-1 isolates, one or two amino acid residues are inserted
into the carboxy-terminal strand of the V3 loop, compared to
the sequence of most HIV-1 strains. The site of these natural
insertions in the V3 stem corresponds precisely to the insertion
site in mutants 3 and 4. Interestingly, mutants 3 and 4 retained
some function in the cell-cell fusion assay, in contrast to most
of the other mutants. The V3 loops of the group O (outlier
group) of HIV-1 are also longer compared to those of most
group M viruses, again due to insertions in the carboxy-termi-
nal strand of the loop (42). Thus, at least some length variation
in the carboxy-terminal V3 stem can be functionally tolerated,
although levels of cell-cell fusion induced by mutants with
changes in this region were lower than that observed for the
wild-type envelope glycoproteins. Naturally occurring HIV-1
strains with V3 insertions may have evolved compensatory
changes in other parts of the envelope glycoproteins.
CD4 binding results in exposure of the V3 loop (49, 59),
even in the context of monomeric gp120 (75). In a crystal
structure of CD4-bound gp120 with an intact V3 region, the tip
of the V3 loop is located ?30 Å away from the gp120 core (38)
(Fig. 6 and 8). Thus, any potential interaction between the V3
tip/stem and the gp120 core in the unliganded conformation is
disrupted upon CD4 binding. This is consistent with the ob-
servation that CD4 binding decreased the binding of gp120 by
the G3-299 antibody, which recognizes a discontinuous epitope
composed of V3 and ?21 sequences (51, 52). Moreover, mu-
tant HIV-1 gp120 glycoproteins (H66A and W69L) that spon-
taneously sample the CD4-bound conformation less than wild-
type gp120 (39; Finzi et al., unpublished) were recognized
more efficiently than the wild-type gp120 by G3-299; con-
versely, a gp120 mutant (S375W) that spontaneously samples
the CD4-bound conformation (83) was recognized less effi-
ciently than wild-type gp120 by G3-299 (Table 1 legend). Com-
bined with a recent study on CD4-induced conformational
changes in the topological layers (“layers 1 and 2”) of the
gp120 inner domain (Finzi et al., unpublished), our observa-
tions allow a more comprehensive picture of the network of
gp120 rearrangements that occur upon CD4 binding (Fig. 10).
FIG. 8. HIV-1 gp120 residues contributing to recognition by the G3-299 antibody. The ribbon diagrams of the F105-bound and CD4-bound
gp120 core plus V3 loop (6, 38) are shown from the same perspective as in Fig. 6. The trimeric axis of the HIV-1 envelope glycoproteins is located
on the left side of each structure, in approximately the vertical orientation (44, 48). In the F105-bound gp120 structure, the V3 loop is disordered
(6). The gp120 residues in which alterations resulted in decreased recognition by the G3-299 antibody (less than 50% of the amount of the
recognition observed for the wild-type HIV-1 YU2 gp120) are colored magenta (51; the present study). In the F105-bound gp120, residues that
contact the F105 antibody (?4 Å) are colored blue (6). Serine 375 also contacts the F105 antibody (6).
3158 XIANG ET AL.J. VIROL.
CD4-induced conformational changes in the V3 loop might
alter the gp120-gp41 interaction and help prime gp41 for sub-
sequent steps in the membrane fusion process.
Current models of HIV-1 gp120-CCR5 binding propose two
critical points of contact: (i) between the gp120 V3 base-bridging
sheet and the CCR5 N terminus and (ii) between the V3 tip and
the body of CCR5 (12–14, 23, 24, 37, 38). The hydrophobic patch
in the V3 tip may interact with the hydrophobic pocket thought to
be formed by the membrane-spanning helices of CCR5 (19, 40,
54, 62). Although the stem that separates the V3 base and tip can
tolerate both sequence variation and conformational flexibility
(38, 42), changes in length appear to be more disruptive of che-
mokine receptor binding. All of the V3 insertions studied herein
resulted in significant decreases in CCR5 binding. Variation in
the length or conformation of the V3 stem presumably interferes
with the precise spatial relationship of the two CCR5-interactive
moieties on gp120, precluding high-affinity binding. This interpre-
tation is consistent with the ability of a mutant CCR5 receptor
with an extended N terminus to compensate partially for some of
the V3 loop insertions.
Future studies should allow a more detailed understanding
of the structural relationships involving the V3 loop in the
unliganded HIV-1 envelope glycoprotein trimer, and the con-
tribution of the V3 loop to receptor-induced conformational
transitions leading to HIV-1 entry.
We thank Yvette McLaughlin and Elizabeth Carpelan for manuscript
preparation. We thank Michael Fung (Tanox) for the G3-299 antibody.
This study was supported by NIH grants AI24755, AI39420, and
AI40895; by a Center for HIV/AIDS Vaccine Immunology grant
(AI67854); by a Center for AIDS Research grant (AI24848); by an
unrestricted research grant from the Bristol-Myers Squibb Founda-
tion; by a gift from the late William F. McCarty-Cooper; and by funds
from the International AIDS Vaccine Initiative.
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FIG. 9. The gp120 elements implicated in subunit association in the
unliganded HIV-1 envelope glycoprotein trimer. The CD4-bound
structure of the HIV-1 gp120 core (43) was fitted into the density
derived from cryo-electron tomograms of the unliganded HIV-1 en-
velope glycoprotein trimer (48). The V3 base is colored magenta, and
the other gp120 residues are colored according to the effects of
changes on gp120-gp41 association, as in Fig. 6 (bottom row). In the
upper part of the figure, the viral membrane is at the top and the target
cell at the bottom. The lower part of the figure shows the HIV-1
envelope glycoprotein trimers from the perspective of the target cell.
The positions of the V3 loops (magenta) were approximated by ex-
tending the two beta strands, ?12 and ?13, that lead into the V3 loop
strands (38, 43, 44). The positions of the V1/V2 stems are indicated.
FIG. 10. Model of the conformational changes in the HIV-1 enve-
lope glycoprotein induced by CD4 binding. One of the three subunits
of the HIV-1 envelope glycoprotein trimer is depicted, oriented so that
the viral membrane is at the top of the picture and the trimer axis is
vertical. The gp120 outer domain (OD) is yellow. The HIV-1 gp120
inner domain consists of a ?-sandwich (red) and loops that form three
topological layers (layer 1 [magenta], layer 2 [green], and layer 3
[yellow]). The ?-sandwich and the gp120 N and C termini (cyan) are
the major gp120 elements that mediate interaction with the gp41
ectodomain (32, 55, 84). Layers 1 and 2, as well as the ?20-?21 loop
(blue) and V3 loop (purple), all contribute to stabilizing the interac-
tion of gp120 with gp41 in the unliganded state (left figure). CD4
binding results in the apposition of layer 1 and layer 2; the formation
of the bridging sheet from the ?2, ?3, ?20, and ?21 strands; and the
projection of the V3 loop away from the gp120 core (5, 38, 43, 55; Finzi
et al., unpublished). This rearrangement of gp120 slows the off-rate of
CD4 (Finzi et al., unpublished), promotes chemokine receptor binding
(78), and allows the gp41 ectodomain to undergo additional confor-
mational changes necessary for HIV-1 entry.
VOL. 84, 2010 HIV-1 gp120 V3 LOOP REGULATES TRIMER STABILITY3159
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