JOURNAL OF VIROLOGY, Mar. 2009, p. 2575–2583
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 6
Determinants Flanking the CD4 Binding Loop Modulate Macrophage
Tropism of Human Immunodeficiency Virus Type 1 R5 Envelopes?†
Maria Jose ´ Duenas-Decamp,1Paul J. Peters,1Dennis Burton,2and Paul R. Clapham1*
Program in Molecular Medicine and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01605,1and the Scripps Research Institute, Department of Immunology, IMM2, La Jolla, California 920372
Received 9 October 2008/Accepted 29 December 2008
Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env)
in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic.
Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In
addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with
brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify
additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33)
and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We
first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N)
conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine
in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that
were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to
identify additional determinants. The determinants of macrophage tropism that were identified included
residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3
loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered
env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the
CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4
contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need
to expose critical CD4 contact residues to the immune system.
Human immunodeficiency virus type 1 (HIV-1) requires in-
teractions between viral envelope glycoproteins and cell sur-
face CD4 and coreceptors to trigger fusion and entry into cells.
HIV-1 R5 viruses that specifically use CCR5 as a coreceptor
are those predominantly transmitted (3). Yet, our knowledge
of R5 virus variation in different biological properties is still
limited. In vivo, HIV-1 infection is limited mostly to cells that
express CD4 and appropriate coreceptors. Thus, HIV-1 infects
CD4?T cells, monocyte/macrophage lineage cells, and den-
dritic cells. CCR5 is expressed on each of these cell lineages,
although on T cells, CCR5 is restricted mainly to RO45?
memory cells (1, 16). Early in infection, R5 viruses target and
decimate mucosal CD4?memory T cells (2, 18, 26). R5 viruses
are also predominant in tissues in which monocyte/macro-
phage lineage cells are prevalent, and several reports have
described the presence of highly macrophage-tropic R5 viruses
in brain tissue (11, 12, 20, 22). Previously, we used PCR to
amplify HIV-1 envelope genes directly from patient tissues.
We found that R5 virus envelopes amplified from brain tissue
frequently conferred highly efficient infection of macrophages,
while the majority of those from lymph nodes, blood, and
semen infected macrophages inefficiently (20, 22). Although
those studies examined relatively few infected individuals, they
demonstrated over 1,000-fold variation in macrophage-tropic
HIV-1 R5 viruses. Such variation is likely to have a significant
impact on transmission and pathogenesis.
The envelope (env) determinants of R5 macrophage-tropic
strains are poorly understood. Several studies have shown that
highly macrophage-tropic brain envelopes are able to exploit
low levels of CD4 on macrophages for infection, consistent
with an enhanced interaction between gp120 and CD4. Dunfee
et al. reported that an asparagine residue at position 283 in the
C2 part of the CD4 binding site was present in 41% of enve-
lope sequences from brain tissue specimens of patients with
HIV-associated dementia and in only 8% of envelopes from
non-HIV-associated dementia brain tissue (8). The same study
showed that the presence of N283 (rather than the more con-
served T283) led to an increased affinity of gp120 for CD4,
probably because the side chain of asparagine could more
readily form a hydrogen bond with Q40 on CD4. However, our
previous data showed that N283 is not the only determinant of
macrophage infectivity, since several macrophage-tropic R5
envelopes from brain and semen specimens lacked N283, while
non-macrophage-tropic envelopes from lymph node specimens
carrying N283 were identified (22). Dunfee et al. also reported
that a glycosylation site at residue 386, close to the CD4 bind-
ing loop, influenced exposure of the CD4 binding site and had
an impact on macrophage tropism and sensitivity to the CD4
binding site antibody b12 (9). We have recently confirmed a
role for N386 in resistance to the CD4 binding site monoclonal
antibody (MAb) b12. However, resistance was dependent on
* Corresponding author. Mailing address: Program in Molecular
Medicine, University of Massachusetts Medical School, Biotech II
Suite 315, 373 Plantation St., Worcester, MA 01605. Phone: (508)
856-6281. Fax: (508) 856-4283. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 7 January 2009.
the presence of a proximal residue, R373, which acted together
with N386 to block b12 (7).
Here, we have further investigated envelope determinants of
macrophage tropism by preparing chimeric envelopes from
highly macrophage-tropic and non-macrophage-tropic R5 en-
velopes from brain and lymph node specimens from the same
subject. We show that R5 macrophage tropism is controlled by
several determinants in gp120 that are focused on amino acids
flanking the CD4 binding loop, with a contribution from resi-
dues in the V3 loop.
MATERIALS AND METHODS
Envelopes and molecular constructs for cotransfection and pseudovirion pro-
duction. HIV-1 envelopes described here were derived from subject NA420, a
heterosexual patient with no cognitive impairment and sparse-infiltrate giant-cell
encephalitis who died of AIDS. Samples from lymph node (LN) and frontal lobe
brain tissue were obtained at autopsy and kept frozen at ?80°C. DNA was
extracted as described previously (24). PCR amplification of complete envelopes
was performed as described previously (20). Patient NA420’s envelope genes
were cloned into pSVIIIenv by using conserved KpnI sites (10) and into pBlue-
script for direct mutagenesis.
Cells. 293T cells (6) were used to prepare env-containing (env?) pseudovirions
by transfection. HeLa TZM-bl cells were used to titrate env?pseudovirions and
to evaluate HIV-1 neutralization. HeLa TZM-bl cells express high levels of CD4
and CCR5 and contain ?-galactosidase and luciferase reporter genes under the
control of an HIV long terminal repeat (23, 27). The RC49 cell line is a clone of
the HeLa/CD4/CCR5 cells that express low levels of CD4 (23). Macrophage
cultures were prepared from elutriated monocytes (14), which were provided by
the University of Massachusetts Center for AIDS Research Elutriation Core.
The elutriated monocytes were cultured for 2 days in medium containing mac-
rophage colony-stimulating factor (R&D Systems) and for an additional 5 days
in medium lacking macrophage colony-stimulating factor and then used for virus
infections. Alternatively, macrophages were prepared from blood monocytes by
adherence as described previously (20). On the day prior to infection, the mac-
rophages were washed three times in Versene and incubated at 37°C for 10 min
to loosen cell attachments. Macrophages were then gently scraped off and re-
suspended in RPMI 1640 medium containing 10% human serum, counted, and
seeded into 48-well tissue culture trays at 1.25 ? 105cells per well.
Direct mutagenesis. Site-directed mutagenesis was carried out using a
QuikChange site-directed mutagenesis kit (Stratagene Inc.) using gp160 in
pBluescript plasmids as templates and mutagenic primers to introduce muta-
tions. The presence of the desired mutations was confirmed by sequencing the
insert. gp160 inserts containing the desired mutations were cloned into the
pSVIIIenv vector via KpnI sites.
Production and titration of env?pseudovirions. The env?pNL4.3 construct
and the pSVIIIenv expression vector were used to produce env?pseudovirions
as described previously (20, 22). Briefly, env?pSVIIIenv with env?pNL4.3 was
cotransfected into 293T cells by using calcium phosphate. Cell supernatants
carrying pseudovirions were harvested 48 h after transfection, clarified (1,000 ?
g for 10 min), aliquoted, and stored at ?152°C.
Comparison of env?pseudotype virus infectivity of primary macrophages and
RC49 cells with infectivity of HeLa TZM-bl cells. Pseudovirions carrying mutant
and patient-derived envelopes were titrated with TZM-bl cells, RC49 cells that
express low amounts of CD4 (23), and primary macrophages. TZM-bl and RC49
cells (2 ? 104cells/well/0.5 ml) were seeded into 48-well trays on the day prior
to infection, while macrophages were seeded at 1.25 ? 105cells/well. A 0.1-ml
volume of serially diluted (10-fold dilutions) cell-free virus supernatant was
incubated with cells. Infections were performed in duplicate. After 3 h at 37°C,
0.4 ml of growth medium was added. For infection of HeLa TZM-bl, cells were
fixed 2 days postinfection in phosphate-buffered saline (PBS)-0.5% glutaralde-
hyde at 4°C for 15 min. Cells were then washed twice with PBS-0.1% azide and
once in PBS before being stained with X-Gal (0.5 mg of 5-bromo-4-chloro-3-
indolyl-?-D-galactopyranoside/ml [Fisher Bioreagents Inc.], 3 mM potassium fer-
rocyanide, 3 mM potassium ferricyanide, 1 mM magnesium chloride) for 3 h.
For macrophages and HeLa RC49 cells (which do not have a ?-galactosidase
reporter gene), cells were fixed at 7 and 3 days postinfection, respectively, in a
cold (?20°C) 1:1 mixture of methanol-acetone for 10 min at room temperature.
Cells were then washed once with PBS-0.1% azide-1% fetal bovine serum, and
a 1:1 mixture of anti-p24 MAbs, 38:96K and EF7 (Centre for AIDS Reagents,
United Kingdom), was added. After 1 h at room temperature, the cells were
rinsed twice in PBS-0.1% azide-1% fetal bovine serum, and goat anti-mouse
?-galactosidase conjugate was added at room temperature for 60 min. Cells were
then washed twice with PBS-0.1% azide and once with PBS before being stained
with X-Gal, as described above for HeLa TZM-bl cells. Since env?pseudovirions
are capable of only a single round of replication, we were able to estimate the
number of focus-forming units (FFU) by counting individual or small groups of
blue-stained, infected cells. Numbers of FFUs/ml were then calculated. For
primary macrophages, cells were pretreated with 0.1 ml DEAE dextran (8 ?g/ml)
in growth medium before virus supernatants were added. Infection of macro-
phages was also aided by spinoculation at 1,200 ? g for 40 min (19).
The infectivity values for RC49 cells and macrophages were also estimated as
a ratio of the infectivity of these cells to the infectivity of TZM-bl cells, and ratios
are presented as a percentage of the ratio observed for B33 env?pseudovirions
by using the following formula: (RC49 or macrophage titer/TZM-bl titer for
mutant env?pseudovirions)/(RC49 or macrophage titer/TZM-bl titer for B33?
pseudovirions) ? 100. Error bars in figures were calculated from replicate wells
for individual experiments.
PRO 542 inhibition assays. HeLa TZM-bl cells (0.1 ml) were seeded into
96-well trays at 8 ?104cells/ml on the day prior to infection. Two hundred FFU
of env?pseudovirions was mixed with twofold serial dilutions of PRO 542 in a
50-?l volume. After 1 h of incubation at 37°C, these mixtures were added to
target cells and incubated for an additional 3 h at 37°C. Then, the virus-antibody
mixture was removed, growth medium was added, and infected cells were incu-
bated at 37°C for 48 h. Medium was then removed, and 100 ?l of medium
without phenol red was added. Cells were then fixed and solubilized by adding
100 ?l of Beta-Glo (Promega Inc.). Luminescence was then read with a BioTek
IC50s and correlations. Virus 50% inhibitory concentrations (IC50s) and cor-
relations were calculated using Prism version 4.0c software for Macintosh. IC50s
were calculated by using a nonlinear regression analysis. In cases where inhibi-
tion did not completely eliminate infectivity, IC50s were estimated manually from
an Excel plot. Correlations were calculated by using a two-tailed, nonparametric
Spearman test with 95% confidence limits.
Here, we have investigated envelope residues that determine
macrophage infectivity for a highly macrophage-tropic HIV-1
envelope (B33) derived from brain tissue by comparison with a
non-macrophage-tropic envelope (LN40) from lymph node tis-
sue from the same individual (patient NA420). In the first part
of the study, we identified envelope determinants that confer
infection via low levels of cell surface CD4, using HeLa RC49
cells as a surrogate for infection of macrophages. We then
confirmed that the residues identified modulated infectivity of
The role of N283 in the capacity to exploit low cell surface
CD4 levels for infection. Residue 283 in the C2 part of the CD4
binding site was previously shown to have a strong influence on
the macrophage tropism of R5 envelopes (8). The presence of
the asparagine residue at 283 (instead of the more conserved
threonine) conferred an increase in macrophage infectivity and
the capacity to infect cells via low levels of CD4. N283 also
confers higher affinity for monomeric gp120 binding to soluble
CD4 (sCD4), strongly suggesting that the capacity to exploit
low CD4 levels results directly from an increased env-CD4
affinity (8). Threonine and isoleucine are the most common
amino acids at residue 283 for subtype B viruses. Patient
NA420’s B33 envelope carries N283, while LN40 carries T283.
We first made the following substitutions at residue 283,
N283T and N283I in B33 (B33T and B33I, respectively) and
T283N and T283I in LN40 (LN40N and LN40I, respectively),
and tested the mutants’ capacity to confer infection of HeLa
cells via high and low levels of CD4. All the mutant envelopes
conferred efficient infection of HeLa TZM-bl cells that express
high levels of CD4 (Fig. 1A). LN40N conferred infectivity for
2576DUENAS-DECAMP ET AL. J. VIROL.
HeLa RC49 cells expressing low amounts of CD4 (23) that was
more than 100-fold greater than that of LN40 wild type (wt).
LN40I conferred only a slight increase in RC49 infectivity
compared to that of the LN40 wt. In contrast, the B33T and
B33I mutants infected RC49 cells efficiently and within 10-fold
of the infectivity for the B33 wt. Thus, the introduction of N283
into LN40 confers substantial infectivity for cells expressing
low levels of CD4. However, the reverse substitution, N283T,
in the B33 mutant had only a small effect. Isoleucine at residue
283 had the same effect as threonine, conferring only minor
effects on B33 and LN40 mutant infectivity of RC49 cells.
Similar conclusions were reached when the RC49 infectivity
titers for LN40 and each of the mutants were normalized and
plotted as a ratio of the infectivity observed for the B33 mutant
(see Materials and Methods) (Fig. 1B). These results confirm
that an asparagine at residue 283 can have a profound effect on
the capacity of HIV-1 R5 envelopes to infect cells via low CD4
levels. However, our data also show that the B33 envelope
must carry additional envelope determinants (independent of
N283) that confer infection of cells via low levels of CD4.
Chimeric envelope constructs used to map envelope deter-
minants of low CD4 use. To map B33 envelope determinants
for low CD4 use, we prepared chimeric envelopes with se-
quences from B33 and LN40 (Fig. 2). An alignment of the
gp120 sequences, including those of B33, B13, B42, LN40, and
LN85 from patient NA420, is shown in Fig. 2A. Chimeric
envelopes were constructed by using StuI and Bsu36I restric-
tion enzyme sites that are, respectively, situated just down-
stream from the V2 loop and immediately upstream from the
conserved GDPE site in the CD4 binding loop (Fig. 2A and B).
To construct the chimeras, we exploited an LN40 envelope
construct that carried the gp41 sequences of B33 and which we
had shown previously required high levels of CD4 for infection
and which infected primary macrophages inefficiently. From
results with this construct, it was already clear that LN40 de-
terminants that conferred a requirement for high CD4 levels
were located in gp120. In addition, all chimeras were prepared
with a threonine residue at 283, since the presence of N283
alone substantially increased the capacity of LN40 to use low
levels of CD4 for RC49 infection (Fig. 1).
Pseudovirions carrying chimeric envelopes were tested for
infection of HeLa TZM-bl and RC49 cells. All the chimeric
envelopes conferred high levels of infectivity for HeLa TZM-bl
FIG. 1. Role of N283 for R5 macrophage tropism. (A) Infectivity
titers of B33, LN40, and mutant envelopes for HeLa TZM-bl cells that
express high levels of CD4 and CCR5 and for RC49 cells that express
low levels of CD4. (B) RC49 infectivity evaluated as ratios with
TZM-bl cell infectivity and then assessed as a percentage of the ratio
for the B33 wt.
FIG. 2. (A) gp160 amino acid sequence alignment for NA420 env.
StuI and Bsu36I restriction sites (vertical lines) used to prepare chi-
meric envelopes occur after residues Q203 and S364, respectively.
Horizontal lines indicate the variable loops. Note that the residue
numbering for envelopes in this figure does not precisely follow
HXBc2 numbering, which is used throughout the text. Thus, LN40
residues H308, F315, R349, N354, I359, N361, Q362, P363, R372, and
N385 are described in the text using HXBc2 numbering as follows:
H308, F317, R350, N355, I360, N362, Q363, P364, R373, and N386,
respectively. (B) Chimeric B33/LN40 envelopes constructed for map-
ping determinants of macrophage tropism.
VOL. 83, 2009 HIV-1 R5 ENVELOPE DETERMINANTS OF MACROPHAGE TROPISM2577
cells (not shown). The Stu chimera contains LN40 gp120 but
carries a segment of B33 gp120, which includes the V1V2
loops. This chimera conferred only a modest increase in RC49
infectivity compared to LN40 (Fig. 3A), indicating that the
V1V2 loops do not play a major role in determining infection
via low CD4. The Bsu-T chimera infection of RC49 was mod-
estly reduced compared to that by the B33T mutant, implicat-
ing an env region stretching from the CD4 binding loop to the
gp120 C terminus for infection via low CD4. However, the
Stu-Bsu chimera showed a more substantial reduction in RC49
infection, this time implicating a region which includes the V3
loop and the N-terminal flank of the CD4 binding loop.
In summary, two regions of gp120 (from LN40) reduced B33
infection of RC49 cells via low CD4 levels. A region that
included the V3 loop and the N-terminal flank of the CD4
binding loop conferred the largest reduction in infection of
RC49. However, a smaller reduction in RC49 infectivity was
conferred by a gp120 region that included a conserved GDPE
motif of the CD4 binding loop and C-terminal sequences up to
the junction with gp41.
The role of the Stu-Bsu fragment in RC49 infection via low
CD4 levels. We next investigated in more detail the role of the
Stu-Bsu region in the infection of RC49 cells via low CD4
levels. This region includes the V3 loop and the N-terminal
flank of the CD4 binding loop and contains 11 amino acid
differences between B33 and LN40, including the N283T
change for LN40 in the C2 CD4 binding site (Fig. 2A). It
should be noted that the numbering system for envelope res-
idues used throughout the text is based on that of HXBc2 and
is not precisely the same as shown in Fig. 2A for envelopes
from patient NA420 (see Fig. 2 legend).
Since all the chimeras and mutants described carry T283
(unless specifically stated), this residue is not involved in the
differences in RC49 infectivity described below. Of the other
10 residues in this region that were different between B33 and
LN40, we were tentatively able to rule out the G350R and
K355N substitutions in LN40, since LN85 (a second non-mac-
rophage-tropic envelope from patient NA420) carried the cor-
responding B33 residues, R350 and N355, at these positions
(Fig. 2A). We therefore constructed B33 envelope mutants
that carried four substitutions on the flank of the CD4 binding
loop (INQP), four additional upstream substitutions (SHFE),
or all eight substitutions together (SHFE-INQP) and tested
their capacity to confer infection of RC49 cells. The SHFE
substitutions include S291 (which introduces a potential N-
linked glycosylation signal into the LN40), H308, and F317
residues within the V3 loop and E335.
Both the SHFE and the INQP B33 mutants conferred mod-
est reductions in RC49 infection. All eight substitutions (SHF
E-INQP) together conferred the largest reduction in RC49
infectivity, to levels close to those conferred by the Stu-Bsu
chimeric envelope (Fig. 3B).
FIG. 3. Identification of gp120 regions responsible for infection of RC49 cells via low CD4. (A) Chimeric env constructs made from B33 and
LN40 env. (B) B33, LN40, and mutant B33 env carrying SHFE, INQP, and SHFE-INQP substitutions. (C) B33, LN40, and mutant B33 env carrying
R373 and N386 substitutions in combination with SHFE-INQP substitutions. (D) The effect of residues on the N-terminal flank of the CD4 binding
loop in combination with downstream residues and determinants in the V3 loop on RC49 infection. (E) B33, LN40, and minimal B33 and LN40
mutants carrying the minimal number of substitutions associated with infectivity for RC49 cells. RC49 infectivity is presented as ratios, with
TZM-bl cell infectivity assessed as a percentage of the ratio for B33 (see Materials and Methods). All chimeric and mutant envelopes carried T283
(with the exception of the LN40 N-NL-KP-KD mutant) to eliminate the effects of N283 on RC49 infection.
2578 DUENAS-DECAMP ET AL.J. VIROL.
We investigated which residues on the N-terminal flank of
the CD4 binding loop affected RC49 infection. Of these resi-
dues, the P at residue 364 conferred the maximum reduction in
RC49 infectivity, while Q363 conferred a more modest reduc-
tion (see Fig. S1A and S1B in the supplemental material).
Several mutants carrying two substitutions in this region were
therefore also tested. Replacement of B33 PS at residues 363
and 364 with QP also resulted in a reduction in infectivity of
RC49 cells, confirming a role for these two residues in the
infection of cells via low CD4 levels.
We also evaluated the effect of substitutions upstream from
the CD4 binding loop flank using the INQP mutants that
carried one or two of the upstream substitutions. Results show
that F317 in the V3 loop conferred the greatest reduction in
infectivity in conjunction with INQP (see Fig. S1C and S1D in
the supplemental material). However, the effect of F317 was
enhanced when H308 was also present.
Together, these results show that Q362 and P363 on the
N-terminal flank of the CD4 binding loop confer a reduction in
infection of RC49 cells via low CD4 levels. However, this
reduction is increased by two further substitutions in the V3
The role of the gp120 fragment, from the BsuI site to the
start of gp41, including N386, in the infection of RC49 cells via
low CD4 levels. The Bsu-T chimera consists of B33 gp120
carrying LN40 sequences from the CD4 binding loop to the
end of gp120 and confers a modest reduction in RC49 infec-
tivity compared to that of B33T (Fig. 3A). This chimera carries
the LN40 residue R373 and a potential glycosylation signal at
N386, which, as we previously reported, conferred resistance to
the CD4 binding site (CD4bs) MAb b12 (7). We first tested an
LN40 N386A mutant that eliminated the potential glycosyl-
ation site at this position. This mutant showed no increase in
RC49 infectivity compared to the LN40 wt, indicating that the
glycan at N386 does not have a major impact on tropism on its
own. We next evaluated whether the presence of R373 and/or
the potential glycosylation site at N386 acted in combination
with the upstream SHFE-INQP substitutions to reduce B33
infection of RC49 cells via low CD4 levels. Figure 3C shows
that residue R373 or N386 conferred only minimal shifts in
RC49 infection when combined with the SHFE-INQP substi-
tutions. However, when R373 and N386 were both combined
with the SHFE-INQP substitutions, a more substantial reduc-
tion in RC49 infection was observed, with infectivity levels
close to those of the background and LN40 infectivity (Fig.
3C). These results suggest that residues R373 and N386 com-
bine with residues on the N-terminal flank of the CD4 binding
loop to impact the capacity to exploit low CD4 for infection.
We sought to confirm this conclusion by constructing and eval-
uating B33 mutants that carried only the critical substitutions
identified above. Thus, we prepared the B33 HF-QP and
QP-RN mutants together with the B33 HF-QP-RN mutant
that contains all six residues identified as those involved in
RC49 infectivity. Figure 3D shows that the HF-QP and QP-RN
mutants conferred a modest reduction in RC49 infectivity
compared to those of B33 or B33T. However, when all six
substitutions were combined in the HF-QP-RN mutant, only
background RC49 infectivity was observed at levels similar to
that of LN40. These results confirm that these six residues act
together to confer the loss of RC49 infectivity observed for
Minimal B33 and LN40 mutants with switched tropism phe-
notypes. The data presented above have suggested that a series
of residues in the V3 loop and on either side of the CD4
binding loop are those critical for the tropism differences ob-
served for the B33 and LN40 mutants. We next prepared LN40
mutants carrying the minimal number of reciprocal substitu-
tions associated with the capacity to infect RC49 cells via low
CD4 levels. The first mutant was constructed with a threonine
at residue 283 to avoid the effects of N283 on tropism. Thus, we
prepared an LN40 (NL-PS-KD) mutant carrying H308N,
F317L (V3 loop substitutions), Q363P, P364S (N-terminal
flank of the CD4 binding loop), R373K (C-terminal flank of
the CD4 binding loop), and N386D (start of the V4 loop). This
NL-PS-KD mutant infected RC49 cells at levels similar to
that of B33T, while an additional NL-PS-KD LN40 mutant
that also carried N283 conferred levels of RC49 infectivity that
were similar to that observed for the B33 wt (Fig. 3E). In
comparison, LN40 and B33 HF-QP-RN mutants conferred low
or background levels of RC49 infection. These observations
strongly indicate that the six residues identified act together to
modulate the capacity of B33 and LN40 to exploit low levels of
CD4 for infection.
The ability to exploit low CD4 levels on RC49 cells corre-
lates with macrophage tropism. All the data presented so far
were derived from experiments that evaluated the infectivity of
different envelope mutants and chimeras on RC49 cells, a
clone of the HeLa cell line that expresses low amounts of CD4
(23). We next tested a panel of the chimeric and mutant en-
velopes for their capacities to confer infection of primary mac-
rophages to confirm that infectivity for RC49 cells is a reliable
indicator for macrophage tropism (Fig. 4). We tested the in-
fectivity of three different batches of macrophages from differ-
ent donors. Data from the macrophage batch that showed
highest sensitivity to B33 are presented. The infectivity data for
other macrophage batches followed the same pattern as that
presented in Fig. 4, except for differences in overall titers. In
addition, macrophage infectivity data generally followed the
same pattern as that recorded for RC49. Thus, B33T conferred
a modest reduction in macrophage infectivity compared to that
of the B33 wt, while LN40N conferred a 1,000-fold increased
infectivity compared to that of the LN40 wt (Fig. 4A). The
Stu-Bsu chimera resulted in about a 100-fold decrease in mac-
rophage infectivity compared to the B33T mutant, while infec-
tivity of the Bsu-T mutant was reduced more modestly. Both
the SHFE and the INQP mutants showed about a 5- to 10-fold
reduction in infectivity for macrophages. However, when these
eight substitutions were combined, macrophage infectivity was
reduced at least 100-fold, a more substantial reduction than
that of the INQP mutant and close to that of LN40 infectivity.
This substantial reduction in macrophage infectivity by the
SHFE-INQP mutant meant that B33 mutants carrying addi-
tional substitutions downstream from the CD4 binding loop,
e.g., R373 and N386, further reduced macrophage infectivity
only marginally. We subsequently confirmed this observation
by testing a B33 T283 HF-QP mutant, which conferred back-
ground levels of macrophage infection similar to the LN40 wt
(not shown). The minimal B33 (B33 HF-QP-RN) mutant also
conferred background macrophage infectivity similar to the
VOL. 83, 2009HIV-1 R5 ENVELOPE DETERMINANTS OF MACROPHAGE TROPISM2579
LN40 mutant, while (in contrast) the reciprocal LN40 (LN40
NL-PS-KD) minimal mutant conferred substantial infectivity
for macrophages (Fig. 4B). Finally, incorporation of N283 into
the LN40 minimal mutant further boosted macrophage infec-
tivity to levels similar to that of the B33 wt (Fig. 4B). The
infectivity of this panel of B33 and LN40 mutants for macro-
phages showed a highly significant correlation with infectivity
measured with RC49 cells (P ? 0.0001).
In summary, our data show that amino acids on the flanks of
the CD4 binding loop in combination with residues in the V3
loop are important for macrophage tropism of R5 envelopes.
However, residues on the N-terminal flank of the CD4 binding
site confer the most significant effects.
Sensitivity to inhibition by PRO 542 (immunoglobulin G-
CD4). Recently, we reported that HIV-1 R5 macrophage tro-
pism correlated with sensitivity to sCD4 and to PRO 542, an
immunoglobulin G-CD4 construct that is tetrameric for CD4
D1D2 domains (21). Thus, pseudovirions carrying highly mac-
rophage-tropic envelopes were sensitive to inhibition by sCD4
and the more potent PRO 542, while non-macrophage-tropic
R5 envelopes were less sensitive or were resistant. These ob-
servations are consistent with R5 macrophage tropism being
conferred by an increase in env-CD4 affinity. To provide more
information about whether B33/LN40 residues, identified as
macrophage tropism determinants, may also have an impact on
env-CD4 affinity (or act via alternative mechanisms), we tested
their effects on the sensitivity to inhibition by PRO 542. Thus,
the B33T mutant showed approximately eightfold increased
resistance to PRO 542 compared to that of the B33 wt (Fig.
5A). In contrast, while the LN40 wt was resistant to PRO 542,
the LN40N mutant was sensitive, although it was still more
than 30-fold more resistant than B33 wt. These observations
reflect the shifts in macrophage and RC49 infectivity observed
with the same envelopes and also indicate that additional en-
velope determinants (over and above residue 283) must mod-
ulate PRO 542 sensitivity.
We next investigated the B33 and LN40 mutants, each car-
rying six reciprocal substitutions that were shown to modulate
the infectivity of RC49 cells. Thus, the B33 HF-QP-RN mutant
(with T283) was as resistant to PRO 542 as LN40, while the
LN40 NL-PS-KD mutant was as sensitive as B33T. These ob-
servations indicate that these minimal mutants fully modulated
PRO 542 sensitivity and resistance, as well as macrophage
tropism (as described above).
We also evaluated the PRO 542 sensitivity of all other B33
and LN40 mutants and chimeras used for mapping the B33/
LN40 determinants of macrophage tropism (not shown). PRO
542 sensitivity correlated with the infectivity of RC49 cells (P ?
0.0001) and macrophages (P ? 0.0005). These results are con-
sistent with macrophage tropism determinants impacting di-
rectly on env-CD4 interactions.
Here, we have mapped envelope determinants of macro-
phage tropism by using two R5 envelopes that were amplified
directly from patient tissues without culture (20). Patient
NA420’s B33 was derived from brain tissue and showed high
macrophage tropism, while LN40 from lymph node tissue in-
fected macrophages inefficiently. Macrophage infectivity was
previously shown to correlate with the capacity to infect cells
via low levels of CD4 (20), while Dunfee et al. reported that an
asparagine at residue 283 in the C2 part of the CD4 binding
site conferred a higher affinity for gp120-CD4 binding (8).
Nevertheless, our previous studies implicated additional un-
known determinants (20, 22), which we sought to identify in
the study reported here. Thus, determinants on the flanks of
the CD4 binding loop and on the V3 loop were determined to
modulate macrophage infection.
The CD4 binding loop is thought to be the most exposed and
perhaps the first part of the CD4bs contacted by CD4 during
HIV entry (4). The substitutions identified on the flanks of the
FIG. 4. Macrophage infectivity for a panel of B33 and LN40 mu-
tants. (A) Macrophage infectivity of B33, LN40, and mutant envelopes
for primary macrophages. (B) Macrophage infectivity for B33 and
LN40 minimal mutants. Infectivity was evaluated as described in Ma-
terials and Methods.
FIG. 5. PRO 542 sensitivity of B33, LN40, and mutants.
2580 DUENAS-DECAMP ET AL.J. VIROL.
CD4 binding loop are either directly adjacent to CD4 contact
residues or in close proximity (Fig. 6 and 7). We predict that
these residues alter the exposure of CD4 contact residues on
this loop, thus allowing better access to CD4 and a correspond-
ing increase in affinity for B33. The affinity of the trimeric
envelopes for CD4 is not straightforward to evaluate (5). Here,
we evaluated the sensitivity of infection to PRO 542, a tet-
rameric soluble CD4 construct based on immunoglobulin G.
An increased affinity of the envelope for CD4 would be ex-
pected to result in increased sensitivity to PRO 542, although
it is possible that PRO 542 inhibition may be conferred by
additional mechanisms. Nevertheless, the B33T and LN40N
mutants showed strong shifts in PRO 542 sensitivity toward
resistance and sensitivity, respectively (compared to their wt
counterparts), as expected for envelopes with altered CD4
binding affinities for gp120 subunits on the trimer. Overall, the
capacity of B33, LN40, and a panel of mutants to infect mac-
rophages or RC49 cells via low CD4 levels showed a highly
significant correlation with sensitivity to PRO 542, consistent
with modulation of env-CD4 affinity.
The N-terminal flank of the CD4 binding loop is variable
(Fig. 6), and such residues have been shown to influence
gp120-CD4 affinity (15). These flanking residues are adjacent
to the highly conserved SGGD-E CD4 contact residues on the
apex of the CD4 binding loop. Such variation is consistent with
a major impact on the exposure of these CD4 contact residues
for this region and is suggestive of an immunity-mediated mod-
ulation. Recently, Sterjovski et al. reported that a potential
glycosylation site (N362) in this flanking region conferred an
increased envelope fusigenicity (25). This observation seems
counterintuitive since the presence of a glycan might be ex-
pected to shield the CD4 binding loop and reduce the effi-
ciency of gp120-CD4 interactions. Nonetheless, Sterjovski’s
observations highlight the effect of this region on envelope-
induced fusion. Our results also implicated residues in the V3
loop (H308 and F317 in non-macrophage-tropic LN40) as de-
terminants of R5 macrophage tropism. How these V3 loop
residues impact macrophage tropism is less clear, and it is
possible that they influence a post-CD4 binding event during
entry, e.g., binding to CCR5. However, B33 mutants carrying
these residues were less sensitive to PRO 542, consistent with
a change in env-CD4 affinity (not shown). The V3 loop extends
30 Å from the gp120-CD4 complex (13). However, the position
of the V3 loop on the unliganded envelope is not known, and
it is possible that it lies close enough to the CD4 binding loop
to influence its exposure. Lynch et al. also reported that a
single amino acid change in the V3 loop of a clade C envelope
conferred sensitivity to soluble CD4 inhibition (17), consistent
with a close association between V3 and the CD4bs.
Our approach in this study was to utilize HeLa RC49 cells as
a surrogate for primary macrophages. RC49 cells express low
FIG. 6. The N-terminal flank of the CD4 binding loop is variable.
A sequence alignment of HIV-1 R5 envelopes previously evaluated for
macrophage infectivity (21) is shown.
FIG. 7. In the top panel, the proximity of macrophage tropism-
determining residues flanking the CD4 binding loop to CD4 contact
residues is shown. Residues Q363 and P364 (red) on the N-terminal
flank of the CD4 binding loop are adjacent to CD4 contact residues
(turquoise) on the apex of the loop. Residues R373 and N386 down-
stream from the CD4 binding loop are shown in green. In the bottom
panel, the glycan at N386 is shown in orange. The structures shown are
those of gp120 complexed with CD4 and MAb 17b (15).
VOL. 83, 2009 HIV-1 R5 ENVELOPE DETERMINANTS OF MACROPHAGE TROPISM2581
levels of CD4 (like macrophages) and their use avoids the
variation in sensitivity to HIV infection observed for macro-
phages from different donors. Once the envelope determinants
that modulated infection of RC49 cells via low CD4 levels were
identified, we tested a representative panel of envelopes, in-
cluding B33, LN40, and critical mutants, with primary macro-
phages from several donors. The infectivity data for this panel
of envelopes were similar for primary macrophages and RC49
cells. However, the V3 loop (HF) and CD4 binding loop N-
terminal flank (QP) residues were sufficient to abrogate mac-
rophage infectivity for B33, without a contribution from down-
stream residues (R373 and N386) which were required to
maximally reduce infectivity for RC49 cells. The mechanistic
basis for this difference between macrophage and RC49 infec-
tion is unclear but presumably relates to subtly different ex-
pression levels of CD4 and/or CCR5 in different cell surface
environments. Nonetheless, the levels of infectivity for primary
macrophages correlated tightly with those for RC49 cells (P ?
0.0001). Finally, infectivity titers determined with different
macrophage batches varied (reflecting donor variation in sen-
sitivity to infection), although the overall pattern of infectivi-
ties remained the same, with B33 as the most macrophage-
tropic and LN40 as the least.
Our data also provide further information on the role of
N283 in macrophage tropism. A T283N substitution in LN40
conferred substantial levels of macrophage infection. The pres-
ence of asparagine at residue N283 thus overrules the involve-
ment of additional residues identified here (in the V3 loop and
on the flanks of the CD4 binding loop) in macrophage tropism.
This could be due to the direct effect of N283 on g120-CD4
binding via the introduction (or improved stabilization) of a
hydrogen bond between N283 and Q40 on CD4, as suggested
by Dunfee et al. (8). The role of the additional residues iden-
tified here presumably involves the modulation of exposure of
the CD4 binding loop (as discussed above) affecting env-CD4
affinity without directly altering the CD4 contact residues.
Thus, our results indicate that env-CD4 affinity may be mod-
ulated by two distinct mechanisms that involve either a direct
effect on the CD4bs or an indirect effect that may affect its
Recently, we reported the determinants in B33 and LN40
envelopes that affect their different sensitivities to the CD4bs
MAb b12 (7). B33 is sensitive to b12, while LN40 is resistant.
In that study, we showed that the LN40 residues Q363 and
P364 on the flank of the CD4 binding loop in combination with
the V3 loop residues H308 and F317 conferred a partial shift
toward resistance to b12 for B33. However, complete B33
resistance was conferred by a combination of R373 and the
glycan at N386. The side chain of R373 and the N386 glycan
appear to combine to fill a proximal cavity on gp120 that is
targeted by the organic ring on the side chain of W100 on b12
(7). Thus, determinants for macrophage tropism and b12 re-
sistance overlap, although different residues have distinct ef-
fects on each phenotype. Our data are consistent with immune
modulation of the identified residues during selection of the
LN40 envelope in the immune environment of the lymph node.
Presumably, the selective force is neutralizing antibodies. In
contrast, in brain tissue, where antibodies are usually restricted
by the blood brain barrier, envelopes like B33 with a more
exposed CD4 binding loop may evolve with an increased sen-
sitivity to neutralization by CD4bs antibodies and an enhanced
capacity to infect macrophages via low levels of CD4. Unfor-
tunately, plasma from subject NA420 is not available to test
directly whether LN40 is more resistant to neutralization than
In summary, we have identified envelope determinants that
modulate R5 macrophage tropism and that have evolved nat-
urally in vivo. The determinants are located on the flanks of the
CD4 binding loop but also involve residues in the V3 loop. We
predict that they modulate exposure of the CD4 binding loop
and accessibility of the CD4bs to CD4. Furthermore, the N-
terminal flank of the CD4 binding loop is variable and such
variation may contribute to a general mechanism for protect-
ing CD4 contact residues on this loop from neutralizing an-
tibodies. Our data have strong relevance for the design of
envelope antigens, which will need to optimally present and
expose residues involved in CD4 binding for the induction
of neutralizing antibodies targeting this critical and con-
This study was supported by NIH grants AI062514, MH064408, and
We acknowledge the University of Massachusetts Center for AIDS
Research (CFAR), the AIDS Research and Reference Reagent Pro-
gram, and the Centre for AIDS Reagents, NIBSC, United Kingdom,
for services and reagents.
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