Functional impact of HIV coreceptor-binding site mutations
Mark J. Biscone, John L. Miamidian, John M. Muchiri1, Sarah S.W. Baik2, Fang-Hua Lee,
Robert W. Doms⁎, Jacqueline D. Reeves⁎
Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA
Received 14 February 2006; returned to author for revision 6 March 2006; accepted 14 March 2006
Available online 21 April 2006
The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We
examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor
susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed
as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used
as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and
CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV
coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779,
which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100
in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to
enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that
reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-
© 2006 Elsevier Inc. All rights reserved.
Keywords: Coreceptor-binding site; Env; HIV; Entry inhibitor; Fusion; TAK-779; AMD-3100; Enfuvirtide; Coreceptor
The envelope protein (Env) of HIV mediates entry into target
cells. Env is composed of a surface subunit, gp120, and a
transmembrane subunit, gp41, which assemble as trimers on the
surface of virions (Center et al., 2002). HIV enters cells
following sequential interactions with the cell surface receptor
CD4 and a coreceptor molecule, usually CCR5 or CXCR4
(Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996;
Doranz et al., 1996; Dragic et al., 1996; Feng et al., 1996;
Trkola et al., 1996; Wu et al., 1996). The coreceptor-binding site
in gp120, together with the third variable loop (V3), mediate
coreceptor binding (Choe et al., 1996; Cocchi et al., 1996;
Kwong et al., 1998; Rizzuto and Sodroski, 2000; Rizzuto et al.,
1998; Speck et al., 1997; Wu et al., 1996). Coreceptor binding
triggers conformational changes in gp41, likely involving
insertion of the hydrophobic fusion peptide into the target cell
membrane, then reorganization of gp41 to bring about fusion
between the cell and viral membranes (Chan et al., 1997;
Weissenhorn et al., 1996).
The coreceptor-binding site in gp120 is centered around an
anti-parallel β-sheet structure, termed the ‘bridging sheet
domain’, that is formed from conserved, discontinuous regions
of gp120 (Kwong et al., 1998; Rizzuto and Sodroski, 2000;
Rizzuto et al., 1998). Mutations in or around the bridging sheet
Virology 351 (2006) 226–236
⁎Corresponding authors. R.W. Doms is to be contacted at fax: +1 215 573
2883. J.D. Reeves, Monogram Biosciences, 345 Oyster Point Blvd., South San
Francisco, CA 94080, USA. Fax: +1 650 624 4132.
E-mail addresses: email@example.com (R.W. Doms),
jreeves@MonogramBio.com (J.D. Reeves).
1Present address: Kenya Methodist University, Department of Applied
Biology, PO Box 267-60200, Meru, Kenya.
2Present address: Dept. of Pathology, UMDNJ, Research Towers R232,
Piscataway, NJ 08854, USA.
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
domain can reduce the efficiency of coreceptor binding (Reeves
et al., 2002, 2004; Rizzuto and Sodroski, 2000; Rizzuto et al.,
1998; Suphaphiphat et al., 2003) as can changes in V3 (Reeves
et al., 2002; Suphaphiphat et al., 2003). In addition, the
sequence of V3 is the major determinant of coreceptor
specificity (Choe et al., 1996; Cocchi et al., 1996; Hoffman et
al., 2002; Hwang et al., 1991; Speck et al., 1997; Wu et al.,
1996). For example, replacement of the V3 loop of a CXCR4
utilizing Env (X4 tropic) with that from a CCR5 tropic virus
(R5) can confer R5 tropism (Hwang et al., 1991).
Of the few Envs that have been examined carefully, R5 Envs
interact with CCR5 with higher affinity (4–15 nM) (Doranz et
al., 1999; Wu et al., 1996) than X4 tropic Envs interact with
CXCR4 (200–500 nM) (Babcock et al., 2001; Hoffman et al.,
2000), with the important caveat being that these measurements
have been done with monomeric gp120. The interplay between
coreceptor specificity and coreceptor-binding efficiency is not
well understood. From mutagenesis studies, a whole range of
CCR5 affinities are compatible with Env fusion and infection
(Reeves et al., 2002, 2004). However, reduced coreceptor
affinity can result in reduced fusion and infection efficiency,
which has consequences for entry inhibition (Reeves et al.,
2002, 2004). For example, reduced CCR5 affinity can increase
susceptibility to entry inhibitors that target CCR5 and to the
fusion inhibitor enfuvirtide (ENF), as a consequence of reduced
rates of fusion (Reeves et al., 2002, 2004). ENF is a peptide
based on the HR2 region in gp41 and competes with HR2 for
HR1 binding to prevent fusion (Greenberg et al., 2004). HR1 is
only exposed following receptor binding (Furuta et al., 1998;
Gallo et al., 2001; He et al., 2003; Melikyan et al., 2000); thus,
there is a window of opportunity between receptor binding and
six-helix bundle formation during which ENF can act.
Therefore, mutations that reduce coreceptor affinity and that
delay fusion kinetics can result in enhanced ENF susceptibility
(Reeves et al., 2002, 2004).
In this study, we compared the impact of mutations in and
adjacent to the bridging sheet domain on CCR5- and CXCR4-
dependent binding, fusion and entry inhibitor susceptibility, as
well as the consequence of these mutations on utilization of two
alternative HIV coreceptors, CCR3 and CCR8. We found that
CXCR4-mediated fusion was more sensitive to coreceptor-
binding site mutations, resulting in a greater reduction in fusion
efficiency compared to equivalent mutations in a CCR5 tropic
Env. Coreceptor-binding site mutations also impacted CCR3
and CCR8 use to varying degrees. Additionally, mutations
increased susceptibility to the CCR5 and CXCR4 inhibitors,
TAK-779 and AMD-3100, but had minimal impact on ENF
susceptibility, consistent with minimal impact on fusion
Mutagenesis of the coreceptor-binding site
Crystallization of a deglycosylated, loop-deleted gp120
protein revealed the presence of two highly conserved regions:
the recessed CD4 binding site and a coreceptor-binding domain,
composed of regions encompassing and adjacent to the bridging
sheet domain (Kwong et al., 1998; Rizzuto and Sodroski, 2000;
Rizzuto et al., 1998). The effects of mutations in the coreceptor-
binding site on Env function have only been examined in a few
Env proteins (Reeves et al., 2002, 2004; Suphaphiphat et al.,
2003). To investigate the role that this region plays in
coreceptor-binding and Env-mediated membrane fusion with
both major and alternative coreceptors, we introduced single or
double point mutations in the bridging sheet and adjoining
regions of the X4 Env HXB and the R5 Env HXB V3BaL.
These Envs are identical save for their V3 loops. Some
mutations were selected based on previous work which showed
that mutations in this region can modulate the affinity of gp120
binding to CCR5 (T123D, K207D, R419D, K421D, P437A,
P438A, G441V, and Q442L) (Reeves et al., 2002, 2004;
Rizzuto and Sodroski, 2000; Rizzuto et al., 1998), while others
were selected based on their location within this domain and
their highly conserved nature (T202D, F423D/I, I423D, I439D,
S440D) (Fig. 1). Mutations were placed in the full-length Envs
as well as in gp120 expression constructs, so that their effects on
fusion and coreceptor binding could be measured.
Effect of coreceptor-binding site mutations on receptor binding
Changes in the coreceptor-binding site could potentially
affect not only coreceptor binding, but CD4 binding as well. To
investigate this, we produced gp120 HXB V3BaL proteins and
examined their ability to interact with CD4 using a cell surface
binding assay. Human 293T cells were infected with a vaccinia
virus encoding T7 polymerase and transfected with gp120-
Fig. 1. Location of mutation sites on HIV-1 gp120. A model of HXB gp120 is
shown,with the bridging sheet region and two-domain solubleCD4 displayedin
blue and yellow ribbon, respectively. The V1/V2 and V3 loop stems are colored
red and orange, respectively. Mutation sites are colored green to indicate
location.All of the mutations are locatedeither within or adjacentto the bridging
sheet domain. This model was rendered utilizing RasMol 2.7.1 and protein
databank file 1GC1; HXB gp120 envelope glycoprotein complexed with sCD4
and induced neutralizing antibody 17b (not depicted) (Kwong et al., 1998).
227M.J. Biscone et al. / Virology 351 (2006) 226–236
encoding plasmids under the control of a T7 promoter and the
media collected 24 h post-transfection. The amount of gp120 in
each sample was measured by an ELISA, and the samples were
diluted as needed such that each contained equivalent amounts
of gp120 as judged by ELISA and by Western blot (data not
shown). To define the linear range of the binding assay, serial
dilutions of wild-type HXB V3BaL gp120 were incubated on
NP2 or NP2/CD4 cells for 2 h at room temperature, after which
the cells were washed, and bound gp120 was detected by
immunostaining and flow cytometry. Once the linear range of
the assay was defined, the entire panel of gp120 mutants was
then tested for the ability to bind to CD4 expressed on the
surface of NP2 cells. The coreceptor-binding site mutations had
no significant effect on the efficiency of CD4 binding (Fig. 2A).
Likewise, when the panel of HXB gp120 proteins was tested, no
significant differences in CD4 binding efficiency were observed
(data not shown).
A similar equilibrium binding assay was used to measure gp120
soluble CD4 (sCD4) to trigger the conformational changes needed
for coreceptor binding. gp120-sCD4 complexes were then added to
T-REx/CCR5 cells, induced to express high levels of CCR5, and
bound gp120 was detected by immunostaining and flow cytometry
(Fig. 2B). We found that HXB V3BaL containing either the P437A
to WT, while T123D, T202D, and S440D showed low levels of
CCR5 binding. The other gp120 mutants exhibited little or no
detectable binding to CCR5 under these conditions. Interestingly,
Q442L exhibited a slight degree of sCD4-independent binding to
CCR5, while parental HXB V3BaL and all of the other mutants
required sCD4 for binding to CCR5. Our results are in good
agreement with the results of Rizzuto and Sodroski (2000) and
Rizzuto et al. (1998) who examined receptor binding of coreceptor-
binding site mutations in the context of YU-2 gp120 core proteins
Fig. 2. Binding efficiencies of HXB V3BaL mutants to CD4 and CCR5. (A) Equivalent amounts of the indicated gp120 proteins were incubated with either NP2 cells
(open bars) or NP2/CD4 cells (closed bars), after which the cells were washed and the amount of bound gp120 detected by immunostaining and flow cytometry
analysis. The amount of HXB V3BaLgp120 usedfor the bindingassay was empirically determined so as to fall within the linear range of the assay, ensuring that either
increased or decreased binding by the mutants could be detected. The amount of HXB V3BaL bound was set to 100%. (B) Equivalent amounts of the indicated gp120
proteins were incubated with T-REx/CCR5 cells in the absence (open bars) or presence (closed bars) of sCD4. After washing, bound gp120 was measured by
immunostaining and flow cytometry analysis. The results in panels A and B are the average + SD of at least three independent experiments with at least two
independent gp120 preparations.
228 M.J. Biscone et al. / Virology 351 (2006) 226–236
with a subset of mutations that we examined in the context of intact
YU-2gp120s (Reevesetal.,2002,2004).Thus,mutations placedin
Env to bind to CCR5 while not affecting CD4 interactions.
that we were unable to quantitate gp120 binding in this equilibrium-
binding assay. This is consistent with other studies that employed
more sensitive binding assays and that showed that T-cell line
adapted HIV-1 gp120 binds to CXCR4 with affinities equal to or
by assaying their AMD-3100 susceptibility (see below).
Effects of coreceptor-binding site mutations on Env-dependent
Although the effects of mutations in the coreceptor-
binding site on interactions between monomeric gp120 and
its receptors have been described (Basmaciogullari et al.,
2002; Reeves et al., 2002, 2004; Rizzuto and Sodroski,
2000; Rizzuto et al., 1998), the effects of coreceptor-binding
site mutations on Env function have only been examined in
detail with one Clade B (Reeves et al., 2002, 2004) and one
Clade C R5 tropic Env (Suphaphiphat et al., 2003). To
analyze the effect of our expanded panel of coreceptor-
binding site mutants on Env function, we first compared
Env expression and processing. We found that the Env
proteins were expressed at similar levels and exhibited
similar levels of gp160 processing and gp120 shedding as
judged by Western blot and flow cytometry analyses (data
not shown). We then determined the effect of coreceptor-
binding site mutations on Env function in a quantitative
cell–cell fusion assay. As shown in Fig. 3A, all of the HXB
V3BaL mutants elicited cell–cell fusion, despite the fact that
some exhibited undetectable CCR5 binding. The T123D,
I439D, S440D, and G441V mutants yielded the lowest
fusion levels at about 60–70% compared to WT. K421D/
F423I, F423D, P438A, and Q442L fused with approximately
70–80% efficiency, while T202D, R419D/F423D, and
Fig. 3. Fusion capabilities of HXB and HXB V3BaL mutant Envs. Relative fusion levels assessed by a cell–cell fusion luciferase reporter assay using QT6 effector
cells expressing the indicated Envs and T7 RNA polymerase, and QT6 target cells expressing CD4 and coreceptor and containing a luciferase reporter gene under the
control of the T7 promoter. Fusion levels are shown for (A) HXB V3BaL mutants and (B) HXB mutants on CD4+ cells (open bars), CD4/CCR5+ cells (black bars),
and CD4/CXCR4+ cells (gray bars). Fusion is expressed as a percent of WT fusion on CD4/CCR5+ cells for HXB V3BaL (A) and fusion on CD4/CXCR4+ cells for
HXB (B). Results represent the average + SD of at least five independent experiments.
229 M.J. Biscone et al. / Virology 351 (2006) 226–236
P437A exhibited essentially wild type fusogenicity. Previous
studies with a subset of the mutations examined here in the
context of YU-2 Env revealed a greater differential impact
on fusion efficiency, ranging from WT levels for P437A and
P438A, intermediate levels of about 25% for T202G and
G441V and markedly reduced levels for K421D and I423S
to approximately 6% of WT (Reeves et al., 2004). Thus, the
effects of mutations in this region of gp120 on fusion
activity via CCR5 can depend on the Env background.
The panel of HXB Env mutants were also examined in a
cell–cell fusion assay to examine the effects of coreceptor-
binding site mutations on CD4/CXCR4-dependent membrane
fusion. In this context, all of the mutations resulted in reduced
fusion compared to WT Env, with T123D and Q442Lexhibiting
the highest level of fusion at approximately 60% of WT (Fig.
3B). Every other mutation showed fusion levels lower than 30%
of WT, with K421D displaying the lowest fusion level at about
10% of WT. Taken together, these results indicate that
coreceptor-binding site mutations can reduce the efficiency of
Env-mediated cell–cell fusion. In the contexts studied here,
mutations were more detrimental on CD4/CXCR4-dependent
membrane fusion than on CD4/CCR5-dependent membrane
fusion, despite markedly reducing the ability of gp120 proteins
to interact with CCR5.
Effect of HXB V3BaL mutations on CCR3 and CCR8 mediated
In addition to CCR5 and CXCR4, several other seven
transmembrane domain coreceptors can support Env-mediated
membrane fusion and virus infection, particularly when
expressed at high levels (Berger et al., 1999). The role of the
bridging sheet domain and adjacent regions of Env in mediating
interactions with these alternative coreceptors is not known. To
investigate this, cells expressing HXB V3BaL Envs were
incubated with cells expressing CD4 and either CCR3 or CCR8,
and the extent of cell–cell fusion was determined. WT HXB
approximately 35 and 20% efficiency, respectively, compared to
CD4/CCR5-positive cells. A range of different activities was
observed for mutant Env fusion via these alternative coreceptors
(Fig. 4). In general, most mutations decreased the efficiency of
CCR3- and CCR8-dependent membrane fusion, with the
exception of T202D, P437A and Q442L for CCR3, and
T202D and P437A for CCR8-dependent fusion. Of the
remaining mutations, some reduced fusion to approximately
the same extent as they reduced fusion on CCR5-positive cells,
including T123D (approximately 60% of WT Env function),
while others reduced fusion to a greater extent, ranging from
about 15 to 50% of WT for CCR3 for S440D and P438A and 20
Q442L. Thus, mutations in the coreceptor-binding site affect the
ability of Env to support membrane fusion that is dependent
upon either the major or alternative coreceptors. However, the
effects of these mutations on CCR3- and CCR8-dependent
membrane fusion were more variable than they were upon
CCR5- and CXCR4-dependent membrane fusion. Additionally,
CCR3, or CCR8 was variable, suggesting that Envs rely on
differential interactions with various coreceptors for fusion.
Inhibition of fusion by a CCR5 antagonist
Several mutations in the coreceptor-binding site reduced the
affinity of HXB V3BaL gp120 for CCR5 to the point that
binding could not be detected in our equilibrium binding assay.
Despite this significant change in coreceptor binding, these
mutants mediated cell–cell fusion as efficiently, or nearly as
efficiently, as the parental HXB V3BaL. This apparent
discrepancy could be explained if the mutations in gp120
affected coreceptor binding to a greater extent in the context of
monomeric gp120 than in the context of the native Env trimer.
Fig. 4. Fusion efficiencies of HXB V3BaL mutants via CCR3 and CCR8. Relative fusion levels via CCR3 and CCR8, assessed by a cell–cell fusion luciferase reporter
assay using QT6 effector cells expressing the indicated Envs and T7 RNA polymerase, and QT6 target cells expressing CD4, coreceptor and a luciferase reporter gene
under the control of the T7 promoter. Relative fusion levels are shown for HXB V3BaL mutants on CD4+ cells (open bars), CD4/CCR3+ cells (black bars), and CD4/
CCR8+cells(graybars). Fusionisexpressedas a percentof HXBV3BaLfusiononCD4/CCR5+cells(set to 100%).Results represent the average+SD of atleastfive
230 M.J. Biscone et al. / Virology 351 (2006) 226–236
We sought to address this possibility indirectly by testing a
subset of the HXB V3BaL mutants for their susceptibility to
TAK-779, a small molecule CCR5 inhibitor. We reasoned that
Env proteins that bind to CCR5 with reduced affinity would
exhibit enhanced susceptibility to TAK-779 as shown previ-
ously (Reeves et al., 2002, 2004).
We found that TAK-779 susceptibility (Fig. 5A) largely
correlated with CCR5 binding (Fig. 2B). The TAK-779
susceptibility of Q442L was similar to HXB V3BaL, suggesting
that Q442L Env binds CCR5 in a WT manner as seen with
Q422L gp120. Furthermore, T202D increased TAK-779
susceptibility by about 3-fold, consistent with the ability of
this mutant Env to bind to CCR5 at 35% of WT gp120. The
gp120 mutants that bound undetectably to CCR5 exhibited
increased susceptibility to TAK-779 from approximately 10- to
25-fold. Slight differences in relative orders obtained in the
binding assay versus those found in the inhibition assay may be
due to the fact that in the binding assay, the monomeric
interaction of gp120 to CCR5 is measured, while in the
inhibition assay, multimeric interactions of trimers interacting
with multiple CCR5 molecules are possible.
Inhibition of fusion by a CXCR4 antagonist
The impaired ability of the HXB coreceptor-binding site
mutants to elicit membrane fusion via CXCR4 (Fig. 3B)
suggested that they would show increased susceptibility to
AMD-3100. This was the case for five of the six point mutations
selected for this study. T123D and T202D were most
susceptible to AMD-3100 inhibition, exhibiting approximately
a 7- to 8-fold reduction in IC50values, while R419D, I423D,
and P438A were only reduced by 2.25- to 3.5- fold (Fig. 5B).
Q442L, on the other hand, showed a slight increase in IC50over
HXB (1.4-fold), which may indicate an enhanced ability to
utilize CXCR4. This was not reflected by an increase in fusion
efficiency, however, as Q442L elicited fusion to only 60% of
Inhibition of fusion by enfuvirtide
Given that the affinity for CCR5 was reduced by many of
these mutations, it might be expected that this would increase
their susceptibility to enfuvirtide since fusion may now proceed
more slowly (Reeves et al., 2002, 2004). However, when
enfuvirtide IC50's were compared across a subset of six R5 and
X4 Env mutant viruses, less than a 3-fold range was noted—
differences that are not likely to be significant (Table 1). We
found this result surprising, since in other Envs reduction in
coreceptor affinity has been associated with reduced fusion
kinetics and enhanced susceptibility to enfuvirtide (Reeves et
al., 2002, 2004). However, when we examined the rate of cell
fusion elicited by these mutants, we found only minimal effects
on fusion kinetics (data not shown), consistent with minimal
differences in ENF susceptibility. Differences were not
observed when cells expressing lower levels of coreceptor
were used. Thus, the effect of reduced coreceptor affinity in the
context of this laboratory-adapted Env had relatively little
impact on fusion kinetics and ENF susceptibility compared to
the effect of equivalent mutations in the context of the R5 tropic
Fig. 5. Entry inhibitor susceptibility of HXB and HXB V3BaL mutants. IC50
values for fusion inhibition by AMD-3100 and TAK-779 were determined in a
cell–cell fusion luciferase reporter assay using QT6 effector cells expressing the
indicated Envs and T7 RNA polymerase, and QT6 target cells expressing CD4,
coreceptor and containing a luciferase reporter gene under the control of the T7
promoter. The IC50value was defined as the concentration of inhibitor needed to
reduce fusion activity by 50%. (A) TAK-779 IC50values for HXB V3BaL
mutants on CD4/CCR5+ QT6 cells. (B) AMD-3100 IC50values for HXB
mutants on CD4/CXCR4+ QT6 cells. Results represent the average IC50
value + SEM of at least four independent experiments.
Fusion inhibition by ENF
Mutation ENF IC50fold changea
ENF inhibition of HXB V3BaL and HXB mutant fusion.
aENF susceptibility of mutant Envs expressed as fold change over WT Env
ENF IC50. Results are derived from at least 3 independent experiments.
231 M.J. Biscone et al. / Virology 351 (2006) 226–236
primary YU-2 Env (Reeves et al., 2002, 2004). As detailed in
the discussion, we speculate that laboratory-adapted Envs may
be triggered more efficiently than at least some primary Envs,
with the result being that reduced coreceptor affinity may not
significantly impact fusion kinetics.
Fusion triggering by soluble CD4
A number of HXB V3BaL mutants mediated cell–cell fusion
as efficiently or nearly as efficiently as the wild-type protein
despite the fact that we were unable to detect binding of their
monomeric gp120 proteins to CCR5 (Figs. 3A and 2B). The
most striking of these, the combination mutant R419D/F423I,
fused as efficiently as the wt protein without binding detectably
to CCR5 in the context of the monomeric gp120-binding assay.
The fact that this mutant could elicit fusion via CCR5
demonstrates its ability to bind to CCR5. Thus, in the HXB
V3BaL native trimer, the consequences of reduced affinity on
fusogenicity can be masked. Alternatively, the coreceptor-
binding site mutations may alter the conformation of Env in a
way that can compensate for reduced affinity, perhaps by
lowering the energy threshold required for Env triggering and
ultimately, fusion. To examine the effect that these mutations
had on CD4 triggering efficiency, we measured the ability of
envelopes to mediate fusion on CD4−/CCR5+ cells in the
presence of increasing amounts of sCD4. Fusion levels in this
assay would also likely be more reliant upon CCR5 binding
affinity. In cell–cell fusion assays using CD4/CCR5+ target
cells, cells are “tethered” together through the interaction of
CD4 and envelope. This “tethering” potentially conceals the
Fig. 6. sCD4 induced fusion of HXB and HXB V3BaL mutants. Relative fusion levels assessed by a cell–cell fusion luciferase reporter assay using QT6 effector cells
expressing the indicated Envs and T7 RNA polymerase, and QT6 target cells expressing coreceptor alone and containing a luciferase reportergene under the control of
the T7 promoter. To assess dose-dependent soluble CD4 (sCD4) triggering of Env-mediated fusion, conventional membrane-bound CD4 was replaced with increasing
concentrationsof sCD4(white—0μg/ml,light gray—0.1 μg/ml, darkgray—1μg/ml,black—5 μg/ml). (A) Fusionof HXB V3BaLon CCR5+cells inthe presence of
5 μg/ml of sCD4 was set to 100%. (B) Fusion of HXB on CXCR4+ cells in the presence of 5 μg/ml of sCD4 was set to 100%. Results represent the average + SD of at
least three independent experiments.
232M.J. Biscone et al. / Virology 351 (2006) 226–236
effects of lowered affinity to CCR5 by holding the cells
together, while enough coreceptor-binding events occur to
allow the cells to fuse. Thus, the sCD4-triggering assay takes
away this bridge, placing an emphasis on the envelope–
coreceptor interaction, and allows examination of ability of the
sCD4-triggered trimer to interact with coreceptor and cause
sCD4 triggered the HXB V3BaL Q442L mutant to fuse with
somewhat better than WT efficiency (Fig. 6A). gp120 binding
and TAK-779 susceptibility (Figs. 2B and 5A) would indicate
that Q422L interacts with CCR5 with WT efficiency, however,
unlike WT Env, this mutant can bind and fuse inefficiently with
CCR5+ cells in the absence of CD4 triggering (Fig. 6A).
Therefore, Q422L may alter the conformation of Env to allow
coreceptor binding in the absence of sCD4 triggering, which
may reflect a reduced requirement of sCD4 to trigger fusion
compared to WT Env. sCD4 triggered near WT fusion of
T202D and P437A mutants (Fig. 6A), consistent with their
CCR5 binding and TAK-779 susceptibility profiles (Figs. 2B
and 5A). P438A, I439D, and S440D exhibited intermediate
levels of fusion following sCD4 triggering, while R419D/
F423I, F423D, and G441V triggered inefficiently. T123D and
K421D/F423I exhibited minimal or no sCD4-induced fusion,
though both utilized CCR5 for fusion in the presence of
membrane bound CD4. The lack of sCD4-induced triggering
for the T123D mutant was surprising since this Env binds
detectably to CCR5 (Fig. 2B). However, this mutant was also
more susceptible to TAK-779 inhibition than would have been
expected (Fig. 5A), indicating that CCR5 interactions of the
trimeric Env may be compromised. Conversely, P438A, I439D
and G441V triggered more efficiently than might have been
expected (Fig. 6A) in comparison to other mutants, from their
undetectable CCR5 binding profiles (Fig. 2B). In the context of
YU-2 Env, the P438A and G441V mutants were also found to
fuse and/or exhibit sCD4-induced triggering more efficiently
than would have been expected from low CCR5 binding
(Reeves et al., 2004).
Inthe contextofHXB,sCD4inducedfusionvia CXCR4 was
reduced for all mutants, consistent with cellular CD4-induced
fusion (Figs. 3B and 6B). Again the Q442L mutant triggered
more efficiently than the other mutants (Fig. 6B) consistent with
reduced AMD3100 susceptibility (Fig. 5B). Additionally, in
contrast to relatively efficient cellular CD4-induced fusion of
been expected, which is consistent with enhanced AMD3100
susceptibility compared to the other mutants tested.
Binding of coreceptors to HIV-1 gp120 is mediated by the
V3 loop and the coreceptor-binding site located in the bridging
sheet of gp120, with the conserved β19 strand being of
particular importance (Reeves et al., 2002, 2004; Rizzuto and
Sodroski, 2000; Rizzuto et al., 1998; Suphaphiphat et al., 2003).
The coreceptor-binding site and the adjoining base of the V3
loop are thought to interact with the amino terminal domains of
the coreceptors (Hartley et al., 2005; Hung et al., 1999; Rizzuto
and Sodroski, 2000; Rizzuto et al., 1998). Sulfated peptides
based on the N-terminal domain of CCR5 bind directly to
gp120, but only after gp120 binds soluble CD4 (Cormier et al.,
2000, 2001; Farzan et al., 2002a, 2000b). Mutagenesis studies
of gp120 also support the idea that the amino terminal domain
of CCR5 interacts with the β19 strand and the base of the V3
loop (Cormier et al., 2001). Interestingly, a portion of the
antigen combining region from a MAb that binds to the
coreceptor-binding site can functionally mimic the N-terminus
of CCR5 (Xiang et al., 2005). Thus, a CCR5 molecule
containing this region in place of the native N-terminal domain
functioned as an efficient coreceptor for R5 virus strains.
Less information is available on role of the coreceptor-
binding site in binding to CXCR4. However, the functional
conservation of the coreceptor-binding site is shown by the fact
that, as in this study, simply switching the V3 loop between an
R5 and an X4 virus can alter coreceptor choice. Thus, the
coreceptor-binding site must be able to interact with either
CCR5 or CXCR4, with coreceptor choice being dictated by the
V3 loop. In addition, alanine substitutions for several residues
in the β19 strand have been shown to reduce gp120 binding and
CXCR4-dependent membrane fusion for both a clade C and a
clade B Env (Rizzuto and Sodroski, 2000; Rizzuto et al., 1998;
Suphaphiphat et al., 2003).
Our mutagenesis studies provide further support that the
bridging sheet, and the β19 strand, are important not only for
as well. In general, the mutations that we introduced universally
suppressed fusion activity that was dependent upon CXCR4,
CCR3, or CCR8. The mutations typically had more modest
is due to the fact that HXB-V3BaL binds to CCR5 with higher
affinity than HXB binds to CXCR4. Thus, for functional effects
loss in binding affinity than does HXB, which already binds to
its coreceptor weakly. The coreceptor-binding site mutations in
HXB-V3 BaL did increase the susceptibility of the resulting
Envs to inhibition by a small molecule CCR5 antagonist, and
they did reduce binding affinity as judged by a cell-surface
gp120-binding assay. We did not identify mutations that had a
profound effect on utilization of one coreceptor, with little effect
on the use of others. Perhaps the manner in which Env engages
the N-termini of various coreceptors is quite similar. Both the
CCR5 and CXCR4 N-terminal domains are negatively charged,
and both contain one or more sulfated tyrosine residues, though
sulfation appears to be more important for efficient utilization of
CCR5 than for CXCR4 (Choe et al., 2003; Farzan et al., 2002a).
Together, our results and previous studies indicate that the
bridging sheet region of gp120 comprises a universal corecep-
tor-binding site that is important for efficient utilization of both
major and minor coreceptors.
The most surprising finding in our study was that mutations
in the coreceptor-binding site, while enhancing susceptibility of
Env to coreceptor antagonists, had little discernable effect on
susceptibility to fusion inhibition by ENF. We have found that
some of the same mutations studied here, when introduced into
primary R5 Env proteins, reduce coreceptor-binding affinity,
233M.J. Biscone et al. / Virology 351 (2006) 226–236
delay fusion kinetics, and enhance susceptibility to ENF
(Reeves et al., 2002, 2004). However, coreceptor affinity is
but one factor that governs fusion kinetics and ENF
susceptibility. For example, a P438A change in the context of
the R5 Env YU-2 markedly reduces binding to CCR5 and
enhances susceptibility to CCR5 inhibitors but has little effect
on fusion extent, fusion kinetics, and ENF susceptibility
(Reeves et al., 2004). Thus, we concluded that this change,
while reducing binding affinity, increases the efficiency with
which this Env is triggered to undergo the fusion-inducing
conformational changes that are induced by coreceptor binding.
This highlights an area of HIVentry about which relatively little
is known—how binding of coreceptor to gp120 induces
structural alterations in gp41. Given the variability in Env, it
would not be surprising if some Envs are easier to ‘trigger’ than
others. Envs that are often referred to as being more ‘fusogenic’
may well fall into this category, perhaps being triggered to cause
fusion by single coreceptor-binding events. In fact, Berger and
colleagues found that HXB is particularly easy to trigger by
receptor binding, since HXB heterotrimers composed of some
subunits that are not competent to bind CD4 and some that have
a defective fusion peptide or mixed trimers of mutant HXB and
wild-type SF162 can cause membrane fusion nonetheless
(Salzwedel and Berger, 2000). Thus, coreceptor binding to
one gp120 subunit in these Env trimers was sufficient to induce
conformational changes in other subunits. Enhanced triggering
may be a general property of laboratory-adapted isolates such as
HXB, where in the absence of immune selection continual
passage on CXCR4-expressing cell lines might be expected to
select for variants that enter cells more quickly. If this
speculation is correct, then reductions in coreceptor affinity in
laboratory-adapted HIV isolates would be expected to have
more modest effects on fusion rates than the same changes
introduced into the context of primary virus isolates. Alterna-
tively, it could be that binding CXCR4 triggers Env more
efficiently than does binding to CCR5. If this is the case, then
some of the changes described here might have differential
effects on fusion kinetics when introduced into an R5X4 Env
protein, depending on whether CCR5 or CXCR4 is introduced
into the target cell. If so, their effects on entry inhibitor
susceptibility, particularly that of ENF, might also be variable
since the potency of enfuvirtide is linked in part to the rate at
which membrane fusion occurs.
Materials and methods
Cell lines QT6, 293T, NP2 (Yamanaka et al., 1993), NP2/
CD4 (Soda et al., 2000), T-REx/CCR5 (Reeves et al., 2002),
U87/CD4/CCR5, and U87/CD4/CXCR4 (Bjorndal et al., 1997;
Deng et al., 1997) were cultured in Dulbecco's modified
Eagle's medium (GIBCO) supplemented with 10% fetal bovine
serum (Hyclone), 100 U/ml of penicillin and 100 μg/ml
streptomycin (GIBCO; DMEM/10/PS). CD4 expression in
NP2/CD4 cells was maintained by adding 1 μg/ml of G418
(Mediatech) to the DMEM/10/PS. T-REx/CCR5 cells required
the addition of blasticidin (5 μg/ml, Invitrogen) and zeocin
(200 μg/ml, Invitrogen) to retain the tet-repressor and ccr5
genes, respectively. High-level CCR5 expression was induced
on T-REx/CCR5 cells by addition of 10 ng/ml doxycycline
(Sigma) to the culture media. CD4 expression was maintained
in the U87 cell lines with 0.3 mg/ml G418, while 1 μg/ml
puromycin (Sigma) maintained CCR5 or CXCR4 expression.
HXB and HXB V3BaL gp160s were cloned into the pSP73
(Promega) expression vectors as previously described (Hoffman
et al., 1998, 1999). Amino acid substitutions T123D, T202D,
K207D, R419D, K421D, I423D, P438A, I439D, G441V, and
Q442L (Fig. 1) were introduced into the HXB backbone
sequence and T123D, T202D, R419D/F423I, K421D/F423I,
F423D, P437A, P438A, I439D, S440D, G441V, and Q442L
(Fig. 1) amino acid substitutions were introduced into HXB
V3BaL using the Quikchange site-directed mutagenesis kit
(Stratagene) according to the manufacturer's instructions. In
order to produce gp120 expression constructs, stop codons were
introduced at the gp120-gp41 cleavage junction in Env for each
of the mutants by site-directed mutagenesis (Quikchange;
Stratagene). Env gene sequences were verified following
The pGEM2 T7-luc expression plasmid was obtained from
Promega. The expression plasmids for CD4, CCR5, CXCR5,
CCR3, CCR8, and pNL-luc-E−have been described previously
(Connor et al., 1995; Deng et al., 1996; Hoffman et al., 1998).
Env receptor-binding assays
gp120 proteins were produced using a vaccinia-T7 poly-
merase-driven expression system (Alexander et al., 1992).
Briefly, 293T cells were transfected with gp120 expression
constructs by calcium phosphate transfection and cells were
infected with a vaccinia virus encoding T7 polymerase (vTF1.1;
Alexander et al., 1992) to boost protein expression via the T7
promoter present in these constructs. Culture media containing
gp120 protein were harvested approximately 24 h post-
transfection. Western blotting was performed to confirm
gp120 production, and an ELISA was utilized to quantify
gp120 protein concentrations as previously described (Reeves
et al., 2002). Briefly, ELISA plates were coated overnight with
10 μg/ml Galanthus nivalis lectin (Vector Laboratories), then
blocked with 2% milk powder in Tris-buffered saline (TBS).
Serial dilutions of gp120 supernatants were then bound to the
plate for 2 h at room temperature alongside a standard set of
serial dilutions of purified HXB gp120 protein. Bound gp120
was detected with an Env-specific rabbit sera followed by a
horseradish peroxidase conjugated anti-rabbit secondary
(Amersham Pharmacia Life Science). Tetramethylbenzidine
(TMB) substrate (Kirkegaard and Perry Laboratories) was then
added, and the colorimetric signal quantitated in a microplate
The relative binding efficiencies of gp120 proteins to CD4
and CCR5 were assessed as described previously (Reeves and
234 M.J. Biscone et al. / Virology 351 (2006) 226–236
Schulz, 1997; Reeves et al., 2002). Equivalent amounts of
gp120 proteins were incubated with NP2 neuroglioma cells and
NP2-CD4 cells to determine relative CD4 binding or T-REx/
CCR5 cells in the presence and absence of sCD4 to determine
sCD4-induced CCR5 binding efficiencies. An Env-specific
rabbit serum was used to detect bound gp120, followed by a
phycoerythrin-conjugated anti-rabbit secondary antibody (Phar-
mingen). The samples were then fixed in paraformaldehyde and
analyzed by flow cytometry. Importantly, the amount of gp120
needed to achieve binding within the linear range of this assay
was empirically determined.
Cell–cell fusion assay
This assay has been previously described in detail (Rucker et
al., 1997). Briefly, “target” QT6 cells were cotransfected with
CD4 and a coreceptor or control expression plasmid as well as a
luciferase reporter gene expression plasmid under the control of
a T7 promoter (pGEM2 T7-luc; Promega). QT6 “effector” cells
were transfected with Env expression plasmids and infected
with a recombinant vaccinia virus expressing T7 polymerase
(vTF1.1) (Alexander et al., 1992). Effector cells were added to
target cells approximately 18 h post-transfection and the cells
allowed to interact at 37 °C for a period of 7–10 h. Quantifiable
fusion occurs following functional envelope–receptor interac-
tions and subsequent content mixing, allowing T7 polymerase
to drive luciferase expression by direct interaction with the T7
promoter (Rucker et al., 1997). Cells were lysed in 1% Triton X-
100 in PBS, luciferase substrate added and luciferase activity
measured in a luminometer.
To determine the ability of soluble CD4 (sCD4) to trigger
cell–cell fusion, variable concentrations of sCD4 (0, 0.1, 1, and
5 μg/ml) were added to mixes of effector and target cells in the
absence of membrane-bound CD4. The TAK-779 and
AMD3100 susceptibility of R5 and X4 tropic Env-mediated
fusion was determined by preincubating target cells with
inhibitors for 30 min prior to addition of effector cells.
To determine the ENF susceptibility of a subset of Env
mutations, variable concentrations of ENF were added to the
target cells prior to addition of the effector cells. Fusion was
then allowed to proceed normally. The IC50for each Env was
then calculated from the inhibition curve produced.
We thank Fred Baribaud for the helpful suggestions. This
work was supported by NIH R01 AI 40880 to R.W.D., and R21
AI 058701 and fellowship 106437-34-RFGN from the
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