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The Amino Terminus of Human CCR5 Is Required for Its Function as a Receptor for Diverse
Human and Simian Immunodeficiency Virus Envelope Glycoproteins
C. Mark Hill,* Douglas Kwon,* Morris Jones,* Craig B. Davis,* Shana Marmon,* Bruce L. Daugherty,³
Julie A. DeMartino,³ Martin S. Springer,³ Derya Unutmaz,*
,
² Dan R. Littman*
,
²
,1
*Skirball Institute of BioMolecular Medicine and ²Howard Hughes Medical Institute, New York University Medical Center,
New York, New York 10016; and ³Merck Research Labs, Rahway, New Jersey 07065
Received February 23, 1998; returned to author for revision April 6, 1998; accepted June 9, 1998
The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses
into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and
have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5
epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent
regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies,
suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with
the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope
glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from
HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data
indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence
requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different
viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed
on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that
Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5
monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all
antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes
cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.
© 1998 Academic Press
INTRODUCTION
Entry of human immunodeficiency virus (HIV) or simian
immunodeficiency virus (SIV) into target cells requires
expression of CD4 and a member of the chemokine
receptor family (Moore et al., 1997). Cellular tropism
maps to the viral Env gene, and all HIV-1, HIV-2, and SIV
Envs identified to this date are able to use chemokine
receptors CXCR4 or CCR5, or both, for viral entry
(Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996;
Doranz et al., 1996; Dragic et al., 1996; Feng et al., 1996;
Simmons et al., 1996; BjoÈrndal et al., 1997; Connor et al.,
1997). Specific HIV and SIV Envs can also utilize addi-
tional members of the chemokine receptor family includ-
ing CCR3, CCR2b, BOB/GPR15, Bonzo/STRL33, GPR1,
V28, and US28 (Alkhatib et al., 1997b; Deng et al., 1997;
Farzan et al., 1997a; Liao et al., 1997; Loetscher et al.,
1997; Pleskoff et al., 1997; Rucker et al., 1997). Chemo-
kine receptors are members of the seven transmem-
brane family of G-protein-coupled receptors and are in-
volved in chemotactic responses of leukocytes (Murphy,
1994, 1995; Schall and Bacon, 1994). The natural ligands,
for example RANTES, MIP-1
a
, and MIP-1
b
for CCR5, are
able to inhibit infection mediated by the cognate receptor
(Cocchi et al., 1995; Bleul et al., 1996; Oberlin et al., 1996;
Samson et al., 1996a). Although the in vivo roles of many
of these receptors in HIV infection is unclear, individuals
homozygous for a null allele of CCR5 are relatively re-
sistant to infection with HIV, indicating an important,
though not absolute, role for this receptor in viral trans-
mission (Dean et al., 1996; Liu et al., 1996; Samson et al.,
1996b). In addition, early in the course of HIV infection,
the virus strains isolated are usually specific for CCR5,
whereas those isolated later have tropism for additional
receptors (BjoÈrndal et al., 1997; Connor et al., 1997; Scar-
latti et al., 1997). These data emphasize a central role for
CCR5 early in HIV infection. Although the majority of
early primary CCR5 tropic viruses are also macrophage
tropic, some viruses identified as CCR5 tropic in in vitro
1
To whom correspondence should be addressed at Howard Hughes
Medical Institute, Skirball Institute of Biomolecular Medicine, New York
University Medical Center, 540 First Avenue, New York, NY 10016. Fax:
(212) 263-5711. E-mail: littman@saturn.med.nyu.edu.
VIROLOGY 248, 357±371 (1998)
ARTICLE NO. VY989283
0042-6822/98 $25.00
Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
357
assays cannot replicate in CD4
1
CCR5
1
monocytes or
macrophages (Cheng-Mayer et al., 1997; Dittmar et al.,
1997). The reasons for this discrepancy in CCR5 tropism
remain unclear and indicate a need to understand in
detail the mechanisms by which CCR5 acts to mediate
viral entry and the role of CCR5 in viral cellular tropism.
Infection by HIV or SIV involves the initial binding of
viral Env to CD4 on the target cell followed by chemokine
receptor-dependent fusion of viral and cellular mem-
branes. There is now considerable evidence that the
mechanism by which CXCR4 and CCR5 mediate mem-
brane fusion events involves direct interactions between
the Envs of HIV or SIV and the extracellular domains of
chemokine receptors. For example, soluble gp120 from a
T cell line tropic Env can form a stable complex with CD4
and CXCR4 on cell surfaces and in solution (Lapham et
al., 1996; Hesselgesser et al., 1997; Ugolini et al., 1997).
In the case of CCR5-tropic Envs, soluble gp120/130 from
HIV-1, HIV-2, and SIV can mediate displacement of iodin-
ated chemokines from cell surface CCR5 in the presence
of cell surface CD4 and can bind directly to cell surface
CCR5 in the presence of soluble CD4 (Trkola et al., 1996;
Wu et al., 1996; Hill et al., 1997). Some of these experi-
ments suggest that soluble Env components can bind
chemokine receptors in the absence of CD4, but it is
clear that CD4 binding to Env greatly increases the
affinity of interaction with CCR5. In addition, one set of
experiments showed that a fragment of CD4 consisting
of the D1D2 domains could displace chemokine from
CCR5 in the absence of HIV gp120, suggesting that the
CCR5 interaction surface of the CD4/Env complex may
involve regions of CD4 as well as Env (Wu et al., 1996).
To further understand the mechanisms by which CCR5
or other chemokine receptors are able to function as
receptors for entry of HIV and SIV, a number of studies
have used mutagenesis and structure-function ap-
proaches to try and define the interactions between Env
and the chemokine receptor. Replacing the amino termi-
nus of murine CCR5 (mCCR5) or human CCR2, which
lack receptor activity for most CCR5 tropic viruses, with
that of CCR5, resulted in infection with some but not all
CCR5 tropic Envs (Aitchison et al., 1996; Rucker et al.,
1996; Alkhatib et al., 1997a; Bieniasz et al., 1997; Doranz
et al., 1997; Edinger et al., 1997; Farzan et al., 1997b; Lu
et al., 1997; Picard et al., 1997). In addition, small trunca-
tions of the amino terminus of CCR5 have been shown to
inhibit infection by some HIV-1 strains (Rucker et al.,
1996). Paradoxically, however, replacement of the amino
terminus of CCR5 with that of CCR2 or mouse CCR5 has
little or no effect on receptor function with any of the
tested virus strains. These and further analyses of other
domains suggest a complex set of interactions between
Envs and extracellular regions of CCR5. However, inter-
pretation of many of these studies has been complicated
by the homology between the fragments of different
chemokine receptors exchanged and the inability to con-
firm the expression and structure of mutant receptor
molecules. Therefore the precise structural requirements
of the extracellular regions of CCR5 in mediating infec-
tion remain unclear.
We have focused on the potential roles of the amino
terminus of CCR5 in expression and folding of CCR5 and
in HIV and SIV receptor function. In this paper, we use a
panel of anti-CCR5 antibodies to analyze the expression
and conformation of a number of amino terminal mutants
of CCR5. We demonstrate that diverse structures at the
amino terminus still allow cell surface expression of
conformationally intact loop regions of CCR5 and that
some of these different amino terminal sequences have
dramatic effects on utilization of CCR5 by an HIV-1 Env
while having little effect on utilization by an HIV-2 or SIV
Env. Complete deletion of the amino terminus has little
effect on the expression and conformation of the loop
regions of CCR5 but leads to a dramatic loss of receptor
activity for the HIV-1, HIV-2, and SIV Envs tested, indicat-
ing a critical role for the CCR5 amino terminus in recep-
tor activity with diverse viral Envs. Additional use of the
anti-CCR5 antibodies to examine the structure of CCR5
on different cell types indicates that CCR5 epitopes
present on 293 cells are not available on CCR5 ex-
pressed on PBMCs. These data suggest that CCR5 may
be subject to different posttranslational modifications or
may adopt specific conformational forms depending on
the cell type in which it is expressed. In addition, binding
of some of the antibodies to PBMCs from an individual
homozygous for a null allele of CCR5 indicate that these
antibodies bind to a molecule which may be related to
CCR5.
RESULTS
Mapping of anti-CCR5 monoclonal antibody binding
to distinct epitopes on CCR5
We initially characterized a panel of anti-CCR5 mono-
clonal antibodies from R&D Systems by determining re-
gions of CCR5 important for their binding. None of the
antibodies bound to 293T cells transiently expressing
CCR1, CCR2, CCR3, or CXCR4, as assessed by FACS
analysis using PE conjugated anti-mouse Ig as a sec-
ond-step reagent (Fig. 1 and data not shown). In contrast,
a similar analysis of cells expressing wild-type CCR5
resulted in a 5- to 50-fold signal over background de-
pending on the antibody used. As a control, we included
the previously described antibody 2D7 (Wu et al., 1997)
(Fig. 1). Initially, to map regions of CCR5 important for
binding of the R&D antibodies, we used chimeric mole-
cules made by exchanging domains of CCR5 with the
equivalent domains of CCR1. The chimeras were tagged
at the carboxyl-terminus with GFP to determine levels of
expression. (Fig. 1A). To provide more detailed informa-
tion on potential antibody interaction sites, small muta-
358 HILL ET AL.
tions were made in each of the extracellular domains of
CCR5 in the context of a molecule containing a HA
epitope tag within the amino terminal domain (Fig. 1B).
The HA epitope was used to confirm expression of each
of the mutant molecules.
The antibodies could be broken down into a number of
groups depending upon the effects that different muta-
tions in CCR5 had on their binding to transfected cells.
The presence of the amino terminal domain of CCR5 was
sufficient for the binding of antibodies 2 and 19 (Fig. 1A),
whereas replacement of the amino terminus with that of
CCR1 resulted in loss of recognition by these antibodies
(Fig. 1A). Binding of both of these antibodies was also
affected by a single amino acid change in the first loop of
CCR5, suggesting that this region could affect the expo-
sure of the N-terminal epitope to these antibodies (Fig.
FIG. 1. Anti-CCR5 antibody binding to mutants of CCR5. Summary of FACS analysis of 293T cells transiently transfected with mutants of CCR5,
incubated with anti-CCR5 antibodies, and stained with PE Goat anti-mouse Ig. Antibodies 2, 17, 19, 23, 29, 31, and 33 are from R&D Systems and
antibody 2D7 is from Leukosite (Materials and Methods). Controls included the intensity of GFP signal or staining with FITC anti-HA. Staining was
graded on the mean fluorescent intensity relative to cells expressing vector alone: 11, 10- to 45-fold; 1, 5- to 10-fold; or 2, background; nd, not
determined. (A) Structure of chimeric constructs is indicated by dark shading for CCR1 fragments and light shading for CCR5 fragments, and the
presence of carboxyl-terminal GFP is as shown. The precise junctions are given in Materials and Methods. (B) Structure of constructs is based on
HA-CCR5 (Materials and Methods), and the amino acids that are exchanged in the mutants are indicated as are their approximate positions within
CCR5.
359AMINO TERMINUS OF CCR5 IN RECEPTOR FUNCTION
1B). In contrast to the first two antibodies, the binding of
antibodies 17, 23, 33, 29, and 31 required the presence of
the loops of CCR5 and was not affected by replacement
of the amino terminus of CCR5 with that of CCR1 (Fig.
1A). This subgroup of five antibodies could be broken
down into two further groups based upon their suscep-
tibility to mutations in CCR5. Mutations in the 59 end of
loop 2 disrupted binding of antibodies 17, 23, and 33,
whereas mutations in the 39 end of loop 2 disrupted
binding of 29 and 31 (Fig. 1B). Further analysis showed
that the presence of loop 2 and loop 3 of CCR5 was not
sufficient for binding of any of these antibodies (Fig. 1A).
Cell surface expression of this construct was confirmed
by the binding of 2D7 which has previously been shown
to bind loop 2 of CCR5 (Wu et al., 1997) (Fig. 1A). These
data indicate that antibodies 17, 23, 33, 29, and 31 either
bind to epitopes that include loop 2 and additional re-
gions of CCR5 or to epitopes that are highly dependent
on the conformation of loop 2.
Substitution of the CCR5 amino terminus affects
receptor activity with some viral envelope
glycoproteins but not others
Envelopes from three diverse CCR5 tropic viruses
were used to generate pseudotyped virus containing an
Env-defective HIV-1 NL4-3 genome incorporating a lucif-
erase reporter gene (Connor et al., 1995). Use of such
pseudotyped virus results in a single round of infection
which can be determined by assaying luciferase activity
in the target cells (Connor et al., 1995).
Envs from strains of HIV-1 (JRFL), HIV-2 (ST24.1), and
SIV
mac239
were used to prepare pseudotyped virus (Ma-
terials and Methods). All three Envs use CCR5 as a
receptor but are unable to use CCR1, CCR2, or CXCR4
(Chen et al., 1997; Hill et al., 1997; Kirchhoff et al., 1997;
Marcon et al., 1997). In addition, HIV-1 JRFL is a macro-
phage tropic virus, whereas HIV-2 ST24.1 and SIV
mac239
are not. The different pseudotyped viruses were used to
FIG. 2. Effect on receptor function and expression of replacing the amino terminus of CCR5 with analogous regions of other chemokine receptors.
(A) Infection of 293T cells transiently expressing CD4 and individual wild-type or mutant chemokine receptors using NL4-3-Luc-R
2
E
2
(HIV-Luc)
pseudotyped with Envs from HIV-1 JRFL, SIV
mac239
, or HIV-2 ST24.1. Cells were harvested 3 days after infection, and luciferase activity was determined
and is expressed in arbitrary units: counts per second (cps). (B) Analysis of the binding of anti-CCR5 antibodies to the 293T cells used in (A). Anti-CCR5
antibodies bound to cells were detected with Goat PE anti-mouse Ig and then analyzed by FACS. FSC, forward scatter. (C) Alignment of the amino
terminal sequences from CCR5, CCR1, CCR2, and CXCR4. Regions of high homology are boxed. Numbers refer to amino acid sequence of CCR5.
360 HILL ET AL.
infect 293 T cells transiently transfected with constructs
expressing CD4 and individual wild-type or chimeric che-
mokine receptor molecules (Fig. 2A). The chemokine
receptor proteins were fused at the carboxyl-terminus
with GFP to allow a relative measure of cellular expres-
sion.
As previously demonstrated, cells expressing CCR5
were infectable by all three viruses, whereas cells ex-
pressing CCR1 were not infected by any of the
pseudotypes (Fig. 2A). In addition, the HIV-2 and SIV Env
pseudotyped viruses were able to infect cells expressing
molecules in which the amino terminus of CCR5 had
been replaced by that of either CCR1 (1555), CCR2
(2555), or CXCR4 (x4555). In contrast, virus pseudotyped
with JRFL Env had a small but reproducible decrease in
the ability to infect cells expressing 1555 and 2555, vs
wild-type CCR5, and was completely unable to infect
cells expressing x4555 (Fig. 2A). Similar results were
obtained with the Env from the BaL strain of HIV-1 (data
not shown).
FACScan analysis of CD4 expression using anti-CD4
antibodies and PE anti-mouse Ig indicated that each set
of transfected cells expressed similar levels of CD4 (data
not shown). Direct FACScan analysis of the cells ex-
pressing the chimeric receptors indicated that there was
only a two- to threefold difference in the level of human
GFP expressed between different sets of transfectants
(data not shown). We also analyzed the expression of the
chimeric receptors using the panel of anti-CCR5 antibod-
ies (Fig. 2B and Table 1). As already shown in Fig. 1,
antibodies 2 and 19 bound to wild-type CCR5 but not to
1555 and also failed to bind to 2555 or x4555 (Fig. 2B and
Table 1). Antibodies 17, 23, 33, 29, and 31 bound to 1555,
2555, and x4555 (Fig. 2B and Table 1). The binding of
each antibody to the different chimeras varied by only
two to threefold, consistent with the level of GFP expres-
sion. In addition, none of the loop-specific antibodies
showed a marked loss of binding to any of the chimeras
relative to their binding to wild-type CCR5, indicating that
the epitopes for each antibody are equally accessible on
the different chimeras. To determine whether the differ-
ences in expression of the chimeras that we observed
could be significant for CCR5 receptor function, we trans-
fected 293T cells with CD4 and different amounts of
CCR5 expression plasmid and infected the cells as be-
fore. When levels of CCR5 expression were decreased
approximately 10-fold, infection was still greater than
100-fold over background and was only up to 5-fold
decreased from infection with high levels of CCR5 (data
not shown), indicating that the differences in expression
level of the different chimeras was unlikely to be signif-
icant for their receptor function. Overall these data indi-
cate that while diverse amino acid sequences at the
amino terminus of CCR5 (Fig. 2C) have little effect on
viral entry mediated by HIV-2 or SIV Envs, different se-
quences can have dramatic effects on entry mediated by
HIV-1 Envs.
Deletion of the CCR5 amino terminus leads to a loss
of receptor function with diverse viral envelope
glycoproteins
To further investigate the role of the amino terminus of
CCR5 in viral receptor function, we made a series of
constructs with progressive deletions in the amino ter-
minus and assayed each of these in 293T cells using
virus pseudotyped with the same Envs as above. These
constructs were based upon wild-type CCR5 without any
GFP at the carboxyl-terminus (Fig. 3A). Removal of 10, 19,
20, and 28 amino acids resulted in a progressive de-
crease in receptor activity (Fig. 3A). After removal of 28
amino acids, no receptor activity was ever detected for
HIV-1 JRFL Env but minimal receptor activity could some-
times be observed with Envs from SIV
mac239
and HIV-2
ST24.1.
To determine whether the decrease in receptor func-
tion was due to a loss of receptor expression on the cell
surface, we analyzed the transfected cells for their ability
to bind the panel of anti-CCR5 antibodies (Fig. 3B, Table
2). The binding was very similar to that observed with the
amino terminally substituted molecules. Antibodies 2
and 19 both bound to wild-type CCR5 but not to any of the
truncated forms of CCR5. In contrast, antibodies 17, 23,
TABLE 2
Binding of Anti-CCR5 Antibodies to Amino
Terminally Truncated CCR5 Molecules
Anti-CCR5 antibodies
Construct 02 19 17 23 33 29 31
CCR1 2222222
CCR5 1111111111111
D10 2 2 11 11 11 11 11
D19 2 2 11 11 nd 11 nd
D20 2 2 11 11 11 11 11
D28 2 2 11 11 11 11 11
D19 Mod 2 2 11 11 nd 11 nd
TABLE 1
Binding of Anti-CCR5 Antibodies to Amino
Terminally Truncated CCR5 Chimeras
Anti-CCR5 antibodies
Construct 02 19 17 23 33 29 31
CCR1gfp 2222222
CCR5gfp 1111111111111
1555gfp 2 2 11 11 11 11 11
2555gfp 2 2 11 11 11 11 11
x4555gfp 2 2 11 11 11 11 11
361AMINO TERMINUS OF CCR5 IN RECEPTOR FUNCTION
FIG. 3. Effect of truncation of the amino terminus of CCR5 on expression and receptor function. (A) Infection of 293T cells expressing CD4 and
individual wild-type or truncated CCR5 molecules using NL4-3-Luc-R
2
E
2
(HIV-Luc) pseudotyped with Envs from HIV-1 JRFL, SIV
mac239
, and HIV-2
ST24.1. The sequence of wild-type and truncated forms of the amino terminal domain of CCR5 are indicated. Cells were harvested 3 days after
infection, and luciferase activity was determined and is expressed in arbitrary units: counts per second (cps). (B) Analysis of the binding of anti-CCR5
antibodies to the cells used in (A). Antibodies bound to cells were detected with goat PE anti-mouse Ig and then analyzed by FACS.
33, 29, and 31 each bound to all of the truncated forms of
CCR5 (Fig. 3B and Table 2). There was some difference
in expression levels, but these were never more than
2.5-fold relative to wild-type CCR5. Analysis of CD4 ex-
pression levels on the transfected cells indicated less
than twofold variation in each case. These data indicated
that the amino terminus was not required for the expres-
sion of CCR5 but that it was required for receptor func-
tion with diverse HIV and SIV envelopes.
Intriguingly, there was a marked difference in receptor
activity between CCR5 truncated by 19 residues and
CCR5 truncated by an additional single cysteine at res-
idue 20. This was particularly obvious with virus
pseudotyped with JRFL Env (Fig. 3A). In addition, there
was a difference in the receptor activity of CCR5 trun-
cated by 19 amino acids (D19) and CCR5 truncated by 19
amino acids and containing a modified sequence be-
tween cysteine 20 and the first transmembrane domain
(D19 mod). This difference was much greater for virus
pseudotyped with HIV-1 JRFL Env than for virus
pseudotyped with HIV-2 ST24.1 or SIV
mac239
Env (Fig. 3A),
indicating that the precise structure of this region is more
important for the HIV-1 Env-mediated infection than for
the HIV-2 or SIV Env-mediated infection.
The marked difference in receptor activity between
CCR5 truncated by 19 residues and CCR5 truncated by
an additional single cysteine at residue 20, especially for
HIV-1 JRFL Env, prompted us to examine directly the role
of this cysteine in CCR5 receptor function. There is a
single conserved cysteine in each of the extracellular
domains of many of the known chemokine receptors.
Biochemical and genetic analysis of the Duffy antigen/
receptor for chemokines (DARC) has suggested that
there is a disulfide bridge between the conserved cys-
teine in the amino terminal domain and the conserved
cysteine in loop 3 (Tournamille et al., 1997). To determine
the potential role of the two analogous cysteines of CCR5
in receptor function, we mutated each one individually
and both together and assayed the mutants for viral
receptor activity. The mutations had no effect on CCR5
cell surface expression nor receptor function assayed
with the same HIV-1, HIV-2, and SIV Envs used in the
above experiments (data not shown), indicating that
these two cysteines and any putative disulfide bond
between them are not required for viral receptor function.
Fusion of CD4 and CCR5 results in a functional
receptor but does not compensate for a deletion
of the CCR5 amino terminus
The decrease in receptor activity of the amino terminal
truncations of CCR5 could be due to a number of effects
including loss of an envelope-specific interaction site on
CCR5, disruption of an interaction between CCR5 and
CD4, or disruption of an intramolecular interaction within
CCR5 that is required for HIV entry to proceed. We
reasoned that if the amino terminal sequence of CCR5
has a key role in interacting with CD4 during Env-medi-
ated fusion, it may be possible to replace this segment of
the chemokine receptor with the CD4 ectodomain. We
therefore made a construct expressing a direct fusion
between the extracellular domains of CD4 and full-length
CCR5 (Fig. 4). Transfection of this construct into 293T
cells rendered them susceptible to infection with virus
pseudotyped with Env from HIV-1, -2, and SIV as used
above (Fig. 4), although the construct showed a de-
crease in activity relative to wild-type CD4 and CCR5.
Staining with anti-CD4 antibodies was threefold lower on
cells expressing the CD4--CCR5 fusion protein than on
cells expressing wild-type CD4 (data not shown). Stain-
ing of cells with the anti-CCR5 antibodies indicated that
the amino terminal specific antibodies 2 and 19 were
unable to bind to the CD4--CCR5 fusion protein, whereas
the rest of the loop-specific antibodies bound well (data
not shown). Additional constructs were made in which
the amino terminus of CCR5 was truncated in an analo-
gous fashion to the CCR5 constructs used in Fig. 3.
Gradual reduction in the amino terminus of CCR5 led to
a complete reduction in receptor activity for the chimeric
FIG. 4. Infection mediated by a fusion of CD4 extracellular domains
to CCR5 and its effect on deletions in the amino terminus of CCR5.
Infection of 293T cells expressing CD4 and wild-type chemokine re-
ceptors or a CD4-CCR5 fusion molecule. The CD4-CCR5 fusion mole-
cule was truncated at the amino terminus by 19 or 29 amino acids as
indicated (see also Materials and Methods). Virus used was NL4-3-
Luc-R
2
E
2
(HIV-Luc) pseudotyped with Envs from HIV-1 JRFL, SIV
mac239
,
and HIV-2 ST24.1. Cells were harvested 3 days after infection, and
luciferase activity was determined and is expressed in arbitrary units:
counts per second (cps).
363AMINO TERMINUS OF CCR5 IN RECEPTOR FUNCTION
molecule. Expression of these constructs on the cell
surface was confirmed by staining with anti-CD4 and
anti-CCR5 antibodies, and the expression levels were all
within twofold of each other (data not shown). These data
indicated that the direct fusion of CD4 to CCR5 was not
sufficient to compensate for a deletion of the amino
terminus but do not rule out the possibility that CD4 may
interact with this region of the chemokine receptor.
Anti-CCR5 antibodies exhibit differential binding
to CCR5 on different cell types
A number of studies have indicated that viruses that
can use CCR5 expressed on certain cell types (e.g., 293T
cells) are unable to use CCR5 on other cell types (e.g.,
macrophages). We therefore investigated the use of anti-
CCR5 antibodies in determining the expression and con-
formation of CCR5 on different cell types. When 293 cells
were stably transfected with CCR5, each of the antibod-
ies bound well to these cells (Fig. 5 and data not shown).
In contrast, when human PBMCs were incubated with
the antibodies, we observed that only antibodies 29 and
31 bound to the lymphocyte population, while antibodies
2, 17, 19, 23, and 33 did not stain any of the primary cells
(Fig. 5 and data not shown). Three-color analysis of fresh
PBMCs revealed that antibodies 29 and 31 bound weakly
to CD4 and CD8 cells, strongly to CD19
1
cells and
strongly to CD14
1
cells (Fig. 5 and data not shown). The
binding of antibodies 29 and 31 was independent of their
isotype, which is the same as that of antibodies 2, 17, and
23. When we used the 2D7 anti-CCR5 antibody from
Leukosite, we found that it bound only to CD4- and
CD8-positive T cells, consistent with previously pub-
lished results (Bleul et al., 1997). The degree of staining
varied on different days and among three different indi-
viduals, but the same pattern of antibody binding was
observed in each case. The binding of R&D antibodies 2
and 19 to CCR5 on 293 cells was consistently lower than
the binding of Leukosite antibody 2D7, and hence the
absence of binding of antibodies 2 and 19 to primary
CD4 and CD8 cells may simply reflect a quantitative
difference in the level of expression of CCR5 on these
cells. In contrast, binding of R&D antibodies 17, 23, and
33 to CCR5 on 293 cells was consistently as great as the
binding of Leukosite antibody 2D7, and yet the R&D
antibodies appear unable to bind CCR5 present on the
primary human T cells.
As a control we also incubated the same antibodies
with PBMCs from an individual who is homozygous for a
null allele of CCR5. We found that, although the Leukosite
antibodies no longer bound to CD4- or CD8-positive
cells, consistent with the absence of CCR5 on these
cells, the R&D antibodies still bound to the same popu-
lations of cells as in wild-type individuals. These data
thus indicate that R&D antibodies 29 and 31 bind to
CCR5 on 293 cells and to a cross-reactive molecule that
is expressed on B cells, macrophages, and, to a lesser
extent, on primary T cells. It remains unclear whether 29
and 31 also bind to CCR5 on T cells and whether the
cross-reactive target molecule present in CCR5-null
PBMC is a related chemokine receptor family member.
Interestingly none of the antibodies showed significant
CCR5-specific binding to primary monocytes from PB-
MCs; these cells may well express CCR5, but it is unde-
tectable under these conditions.
DISCUSSION
The chemokine receptor CCR5 plays a central role in
CD4-dependent HIV infection and acts as a receptor for
viral entry mediated by a diverse range of both HIV and
SIV Envs. We have examined the role of the amino
terminus in the expression and receptor function of
CCR5. Initially we characterized a panel of anti-CCR5
antibodies and determined that they bound to several
different epitopes on CCR5. Subsequently we used these
antibodies in a structure-function analysis of the amino
terminus of CCR5 to ascertain that chimeric and trun-
cated forms of CCR5 were expressed at the cell surface
and remained conformationally intact. Replacement of
the amino terminus of CCR5 with that of CCR1 or CCR2
had no significant effect on cell surface expression or on
infection mediated by an Env from HIV-1 JRFL. Substitu-
tion with the CXCR4 amino terminus also had no effect
on cell surface receptor expression but led to a complete
loss of receptor activity for this HIV-1 Env. In contrast, all
three amino-terminal-substituted CCR5 molecules acted
as receptors for Envs from HIV-2 ST24.1 and SIV
mac239
.
Despite the ability of the HIV-2 and SIV Envs to utilize
CCR5 chimeras with diverse amino terminal sequences,
deletion of the amino terminus of CCR5 led to a dramatic
loss of receptor activity for these Envs, as well as for
HIV-1 JRFL Env. The deleted CCR5 retained weak recep-
tor activity for the HIV-2 and SIV Envs but had no activity
at all for the HIV-1 Env, suggesting that the HIV-2 and SIV
Envs may be less dependent on the intact CCR5 amino
terminus than the HIV-1 Env, although all three viral Envs
clearly require the amino terminus of CCR5 to mediate
efficient viral entry. These data indicate a critical role for
the CCR5 amino terminus in mediating infection with
diverse viral Envs and suggest that different Envs make
distinct and specific interactions with the CCR5 amino
terminal domain.
Incubation of the anti-CCR5 antibodies with 293 cells
expressing CCR5 and PBMCs from CCR51/1 and
CCR52/2 individuals indicated that some CCR5
epitopes present on 293 cells were unavailable on
PBMC. These data suggest that CCR5 undergoes cell
type specific modifications or makes cell type specific
interactions with other molecules, and these modifica-
tions may affect viral Env interaction with CCR5. In ad-
dition, we found that two anti-CCR5 antibodies, whose
364 HILL ET AL.
FIG. 5. Binding of anti-CCR5 antibodies to 293 cells expressing CCR5 and peripheral blood mononuclear cells. Antibodies were incubated at
saturating concentrations with 293 cells expressing CCR5 and binding was detected with goat PE anti-mouse Ig. Fresh peripheral blood lymphocytes
(Materials and Methods) were incubated with the same concentrations of anti-CCR5 antibodies and were stained with goat PE anti-mouse Ig,
TRICOLOR CD4, and FITC CD8, FITC CD19, or FITC CD14. Cells were subsequently analyzed by single- or three-color FACS analysis. Ig, control
purified IgG1 and IgG2 antibodies.
365AMINO TERMINUS OF CCR5 IN RECEPTOR FUNCTION
binding to CCR5 is dependent on sequences in the
C-terminal segment of external loop 2, appear to cross-
react with an unknown molecule on the surface of
PBMCs.
To understand the mechanism by which CCR5 medi-
ates HIV/SIV entry, it will be necessary to define the role
of individual domains in receptor function. We have fo-
cused on the sequence requirements of the CCR5 amino
terminal domain for interactions with envelope glycopro-
tein that result in viral entry. Substitution with amino
terminal domains of related chemokine receptors or trun-
cation of the amino terminus of CCR5 resulted in a loss
of binding of amino terminal specific antibodies but had
little effect on the binding of other antibodies specific for
the loops of CCR5 (Figs. 2 and 4, Tables 1 and 2). The
results thus indicate that the amino terminus of CCR5 is
not required for surface expression of the receptor and
suggest that modification of the amino terminus does not
lead to significant alteration of the conformation of the
external loops of CCR5. However, truncation of the amino
terminus of CCR5 abrogated receptor activity for each of
the diverse envelopes that we used (Fig. 3). The results
suggest that the CCR5 amino terminus makes direct
interactions with the viral Envs and that these interac-
tions are required for subsequent membrane fusion. Pre-
vious studies using the HIV-1 JRFL Env have also indi-
cated that the amino terminus of CCR5 has an important
role in receptor function. For example, if the first 20 or 28
amino acids of CCR5 were used to replace the equiva-
lent regions of CCR1 or -2, partial receptor activity for
HIV-1 JRFL was transferred to these molecules (Rucker
et al., 1996; Doranz, 1997) (C.M.H., unpublished results).
In addition, consistent with a positive role for the amino
terminus of CCR5, we have shown that replacement of
the amino terminus of CCR5 with that of CXCR4 results in
a complete loss of receptor activity for JRFL Env (Fig. 2),
although this result could also be interpreted as CXCR4
making a negative interaction with JRFL Env.
In contrast, there are a number of experiments that
suggest a neutral role for the amino terminus of CCR5 in
receptor function with JRFL and other Envs. When CCR1
or CCR2 amino termini were replaced with that of CCR5
and these chimeras assayed with SIV
mac239
Env, they did
not act as receptors, suggesting the absence of a role for
the CCR5 amino terminus with this Env (Edinger et al.,
1997) (C.M.H., unpublished results). In addition, replace-
ment of the first 20 amino acids of CCR5 with the equiv-
alent regions of CCR1, CCR2, mCCR5, and CXCR4 was
reported to have little effect on receptor activity with JRFL
Env (Doranz et al., 1997), and we have replaced the entire
CCR5 amino terminus with that of CCR1 and CCR2 with
minimal effect on receptor activity with any of the three
Envs that we have tested (Fig. 2).
The apparently contradictory results can be reconciled
if the amino terminal domain in each chimera makes
important interactions with Envs through conserved res-
idues. Amino acids conserved among the amino termini
of CCR1, CCR2, and CCR5 may be important in receptor
function with the three different Envs that we have ana-
lyzed (e.g., amino acids that align with Y14, P19, C20,
K22, K26, A30 of CCR5; Fig. 2C). Amino acids conserved
among all four amino termini used, including CXCR4,
may allow receptor activity with SIV
mac239
and HIV-2
ST24.1 Env (e.g., Y14, P19, C20, K22; Fig. 2C). In contrast
amino acids that are conserved among CCR1, CCR2, and
CCR5 but are different in CXCR4 may be specifically
involved in infection with JRFL Env. Interestingly this only
occurs at amino acids I23, K26, and A30, suggesting that
this region of CCR5 is important for receptor function
with JRFL Env. Consistent with this suggestion, the trun-
cated CCR5 D19 still retains considerable receptor ac-
tivity relative to background, whereas D19 mod, which
contains a modified sequence between C20 and the first
transmembrane of CCR5, has barely detectable receptor
activity for JRFL Env. In other studies, substitution of Ala
for K22 and K26 had little effect on JRFL Env-mediated
cell±cell fusion or entry (Doranz et al., 1997; Dragic et al.,
1998), and therefore further analysis will be required to
determine the significance of individual amino acids be-
yond C20 in viral entry mediated by this HIV-1 envelope.
The only single mutations shown to have a significant
effect on receptor function with JRFL Env are the nega-
tively charged D2, D11, and E18 (Doranz et al., 1997;
Dragic et al., 1998); these residues can be tentatively
aligned with negative charges in the other receptors but
the significance of this alignment remains to be deter-
mined. Single mutations which affect entry mediated by
SIV
mac239
Env include changes of Y10, D11, or Y14 to A
(Farzan et al., 1998), and mutational studies with this and
other viruses suggest a positive role for a number of
residues between the amino terminus and C20 of CCR5
in mediating entry of diverse viral Envs. These recent
reports indicate a dramatic decrease in receptor activity
with a single point mutation (e.g., D11 with HIV-1 JRFL
Env) (Dragic et al., 1998; Farzan et al., 1998). When we
have made related mutations, we observe 5- to 10-fold
decreases in activity (C.M.H and B.D., unpublished re-
sults), but because of the greater dynamic range of our
assay system, we retain levels of infection for HIV-1 JRFL
and SIV
mac239
Env that are 100-fold over background. The
different sets of data are therefore consistent with a role
for residues 1±19 in entry, but the decrease in activity
seen with a further decrease in the length of the amino
terminus indicates, in addition, an important role for the
region between C20 and the first transmembrane do-
main in entry mediated by diverse viral Envs.
In addition to the CD4-dependent Env binding to
CCR5, Wu et al. (1996) reported that the CD4 D1D2
domains compete for chemokine binding to CCR5. The
CD4-gp120 complex may therefore interact with CCR5
through a novel surface formed by both molecules or
through multipoint contacts with CCR5 mediated by both
366 HILL ET AL.
proteins. If the sole function of the CCR5 N terminus in
HIV entry is to interact with CD4, then replacement of this
region of CCR5 with the CD4 ectodomain may be ex-
pected to restore HIV receptor function. However, trun-
cation of the CCR5 N-terminal domain in such a chimera
eliminated receptor function with all envelopes tested
(Fig. 4). The correlation between receptor function of
internally deleted CD4-CCR5 chimeras and N-terminal-
deleted CCR5 further supports a role for the N terminus
in direct interaction with gp120 or a gp120/CD4 complex
and argues against a simple model in which the N-
terminal sequence of CCR5 is primarily required for re-
cruitment of CD4. However, these results do not rule out
a requisite role for an interaction between CD4 and the
amino terminus of CCR5.
The HIV-1 JRFL Env that we have used is from a
macrophage tropic virus, whereas the HIV-2 ST24.1 and
SIV
mac239
Env are from CCR5 tropic viruses that are
unable to infect macrophages. This difference in tropism
correlates with a difference in the susceptibility to re-
placement of the CCR5 amino terminus with that of
CXCR4 or with random sequence C terminal to the con-
served cysteine. A second macrophage tropic Env, from
HIV-1 BaL, was similarly affected by these substitutions
(C.M.H., unpublished results). It will therefore be inter-
esting to see if entry mediated by additional macrophage
tropic HIV Envs is susceptible to replacement of the
amino terminus of CCR5 with that of CXCR4, thus helping
to confirm a specific role for this region in mediating
entry of macrophage tropic viruses.
One explanation for why certain CCR5 tropic viruses
are able to infect macrophages whereas others are not is
that CCR5 adopts a different structure on cells used in in
vitro assays (e.g., 293 cells) and on macrophages. The
R&D anti-CCR5 antibodies 17, 23, and 33 and the Leu-
cosite antibody 2D7 bound equally well to CCR5 ex-
pressed on 293 cells, but only 2D7 bound well to CCR5
on CD4
1
and CD8
1
T cells. This loss of a CCR5 epitope
on human T cells is consistent with a modification of the
structure of CCR5 on different cell types. This change in
structure could be due to covalent modification of CCR5
(e.g., glycosylation) or could be due to an interaction with
another protein which either masks an epitope directly or
stabilizes a CCR5 conformation which masks the
epitopes concerned. Further biochemical analysis will be
required to differentiate these possibilities, and these
antibodies may be useful reagents to help in the identi-
fication of molecules that interact directly with CCR5 in
vivo. Our studies indicate a difference in the structure of
CCR5 on T cells and 293 cells, but additional studies will
be required to confirm the structure and expression of
CCR5 on macrophages and to help determine the func-
tional significance of these structural modifications in
CCR5 both for chemokine binding and for viral infection.
The binding of R&D anti-CCR5 antibodies 29 and 31 to
B cells and macrophages was unexpected since with
fresh PBMCs the reported binding of anti-CCR5 antibod-
ies has been to CD4
1
and CD8
1
T cells (Bleul et al.,
1997). Comparison of the binding of these antibodies to
PBMCs from a CCR51/1 and a CCR52/2 individual
indicates that these antibodies are not binding to CCR5
but are binding to a molecule present at high levels on B
cells and macrophages and at a low levels on T cells.
Similar to the other R&D anti-CCR5 antibodies, antibod-
ies 29 and 31 bind to CCR5 but not to CCR1, CCR2, CCR3,
or CXCR4 expressed on 293 cells (Fig. 1, Tables 1 and 2;
Monica Tsang, personal communication). Mutation of the
carboxyl-terminal region of loop 2 of CCR5 specifically
abrogates binding of both of these antibodies (Fig. 1),
and this region of loop 2 is highly divergent between all
the known chemokine receptors. These data suggest
that these antibodies may be interacting with a novel
chemokine receptor related to CCR5.
Together with reports from other laboratories, our
study demonstrates the complexity of the interaction of
the HIV and SIV envelope glycoproteins with chemokine
receptors. Fundamental differences in CCR5-tropic vs
dual-tropic HIV-1 Envs may determine their relative de-
pendence on interactions with N-terminal and loop se-
quences in CCR5 (Rucker et al., 1996; Doranz et al., 1997;
Edinger et al., 1997; Lu et al., 1997; Farzan et al., 1998).
Similarly, there may be important differences between
HIV-1 Envs and those from SIV and HIV-2, since the latter
appear to be relatively less dependent on specific se-
quences in the CCR5 N terminus. The greater flexibility of
the SIV and HIV-2 Envs may be due, in part, to a require-
ment for them to also interact with BOB/GPR15 or Bonzo/
STRL-33. Furthermore, the ability of CCR5-tropic viruses
to replicate effectively in macrophages may also be de-
pendent on their ability to interact with specific regions of
the chemokine receptors, and this may also be reflected
in the differences observed between the Envs analyzed
in this study. A combination of further structure-function
studies and analyses of postentry events will likely shed
light on this important problem in HIV pathogenesis.
MATERIALS AND METHODS
Plasmids
Plasmids encoding envelope glycoproteins from the
HIV-1 JRFL, HIV-2 ST24.1, and SIV
mac239
have previously
been described (Hill et al., 1997).
Plasmids expressing wild-type chemokine receptors
with green fluorescent protein (GFP) fused to the carbox-
yl-terminus have previously been described (Deng et al.,
1997; Hill et al., 1997). Briefly, the plasmids were made
using the overlap PCR method and cloned into the vector
pBABE. The last amino acid before the stop codon of
each chemokine receptor was fused to a linker se-
quence GSGGTGSGP, which was fused directly to the
initiator methionine of GFP. The chimeric plasmids with
fragments of CCR1, CCR2, CXCR4, and CCR5 were also
367AMINO TERMINUS OF CCR5 IN RECEPTOR FUNCTION
made by the overlap PCR method. The precise junctions
between the chemokine receptors are indicated below
bya*inthepartial junction amino acid sequences from
the interface between ecto-domains and the transmem-
brane domains.
CCR5 junctions
N terminus 39 AAR*LLP
Loop 1 59 PFW*AHY 39 MCQ*LLT
Loop 2 59 IIF*TRS 39 VIL*GLV
Loop 3 59 VLL*LNT 39 QAM*QVT
CCR1 junctions
N terminus 39 GAQ*LLP
Loop 1 59 PFW*IDY 39 MCK*ILS
Loop 2 59 LYF*SKT 39 NLF*GLV
Loop 3 59 TIL*ISV 39 LAV*QVT
CCR2 junction
N terminus 39 GAQ*LLP
CXCR4 junction
N terminus 39 KIF*LPT
CCR5 with the hemagglutinin (HA) epitope tag inserted
14 amino acids from the amino terminus (HA-CCR5) was
a gift from S. Choe and N. Landau (Liu et al., 1996) and
subcloned into the pBABE vector. The mutants in HA-
CCR5 were all made by the overlap PCR method, and the
amino acid sequences exchanged are shown in Fig. 1.
The plasmids for the truncated forms of CCR5 were
made from wild-type CCR5 using overlap PCR to gener-
ate the deletions (Fig. 3). The vector used for these
plasmids is designated pBJ-neo (DeMartino et al., 1994)
and contains a CMV promoter driving expression of the
inserted gene.
Plasmids for expressing CD4 were either pMXT4,
which has been described previously (Deng et al., 1997),
or pBJ-neo-T4.
The plasmid for expression of CD4 fused to CCR5 was
made by overlap PCR and consists of the four extracel-
lular domains of CD4 fused to the amino terminus of
CCR5 starting at amino acid 2 of CCR5 or the amino acid
indicated in Fig. 4.
Antibodies
Antibodies to human CD4, CD8, CD19, and CD14 were
from Caltag. Monoclonal antibodies 2, 17, 19, 23, 29, 31,
and 33 against CCR5 were generously provided by M.
Tsang (R&D Systems). They were generated by immu-
nizing mice with a murine B cell myeloma expressing
human CCR5. Antibody 2, 17, 23, 29, and 31 are IgG2b,
antibody 19 is IgG3, and antibody 33 is IgG1. The anti-
bodies have been characterized as specific for CCR5
and not CXCR4, CCR2, or CCR3. Antibodies to CCR5 from
Leukosite were provided by H. Heath and C. MacKay and
included 5C7 and 2D7,which are IgG2a. The antibodies
were all used in FACS analyses at concentrations that
were saturating for CCR5 on 293T cells.
FACS analysis
Cells were incubated in 50
m
l of saturating amounts of
antibody diluted in PBS/BSA (0.1%) for 30 min on ice and
then washed with PBS/BSA. Samples containing anti-
CCR5 antibodies were next incubated with phycoerythrin
(PE) conjugated anti-mouse antibody (Caltag), then
washed with PBS/BSA. When staining PBMC, the cell
samples were further incubated on ice with mouse IgG
followed by antibodies to CD4 (Tricolor conjugate) and
CD8, CD19, or CD14 (FITC conjugate). Samples were
washed and analyzed on a Becton Dickinson fluorescent
activated cell scanner (FACScan).
Cells
293 and 293T cells were maintained in high glucose
DMEM (Gibco) containing 10% FCS (Atlanta), penicillin
(Gibco), and streptomycin (Gibco). PBMCs were obtained
by venous puncture and were purified by density centrif-
ugation over Ficoll--Hypaque (Pharmacia), washed, and
analyzed immediately by FACS. 293 cells expressing
CCR5 were made as previously described (Hill et al.,
1997). Briefly, 24 h prior to transfection, 3 3 10
6
cells of
the murine leukemia virus amphotropic packaging cell
line Bing (Pear et al., 1993) were plated on 10-cm dishes.
The cells were transfected by a modification of the
CaPO
4
method (Landau and Littman, 1992) with 20
m
gof
the defective retroviral vector pBABE encoding a human
CCR5 cDNA insert (Morgenstern and Land, 1990; Deng
et al., 1996). Forty-eight hours after transfection virus was
harvested from the cells and used to infect 10
6
293 cells
in 10-cm dishes. Forty-eight hours h later cells were
selected with puromycin (Calbiochem) at 1
m
g/ml, and
after 10 days of growth in selective media, the bulk
population of puromycin-resistant cells were incubated
with anti-CCR5 antibody, stained with a phycoerythrin
conjugated anti-mouse Ig (Caltag), and found to be 100%
positive by fluorescent activated cell scanner (FACScan,
Becton Dickinson) analysis.
Transient transfections
293T cells were transiently transfected by a modifica-
tion of the CaPO
4
method (Landau and Littman, 1992).
Briefly, 24 h prior to transfection, 3 3 10
6
cells were
plated on 10-cm dishes. Cells were cotransfected with 10
m
g of PMXT4 or pBJ-neo-T4 and 10
m
g of individual
plasmids containing inserts for wild type and modified
chemokine receptors. Cells were harvested 24 h after
transfection and replated for subsequent infection with
pseudotyped virus at 48 h after transfection.
368 HILL ET AL.
Pseudotyped virus infection assays
NL4-3-Luc-R
2
E
2
(Connor et al., 1995) virus stocks
pseudotyped with different Envs were generated by
transfecting 293T cells with 10
m
g of pNL4-3-Luc-R
2
E
2
and 10
m
g of envelope expression plasmid. Virus-con-
taining supernatants were harvested 48 h later, clarified
of contaminating cell debris, and frozen at 280°C. Vi-
ruses were quantified by p24 ELISA (Cellular Products
Inc.). Target 293T cells were plated in 24-well plates at
5 3 10
4
per well. Cells were infected 24 h after plating
with 50 ng p24 of luciferase reporter virus in 0.5 ml per
well. After 16 h, 1 ml of fresh medium was added to each
well, and 48±60 h later the cells were harvested and
assayed for luciferase activity using the Promega lucif-
erase assay kit and a Wallac Microbeta 1450 Counter.
ACKNOWLEDGMENTS
We thank Drs. Monica Tsang and Charles Mackay for their generous
gifts of antibodies. This work was supported by NIH grant AI33303 to
D.R.L. D.R.L. is an Investigator of the Howard Hughes Medical Institute.
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