structural determinants of the anti-hiv activity of a ccr5 antagonist
TRANSCRIPT
Structural determinants of the anti-HIV activity of a CCR5 antagonist
derived from Toxoplasma gondii
Felix Yarovinsky1, John F. Andersen2, Lisa R. King ,3 Patricia Caspar1, Julio Aliberti1,4,
Hana Golding3#, Alan Sher1#*
1Immunobiology Section, Laboratory of Parasitic Diseases and 2Laboratory of Malaria and
Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, MD, USA
3Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and
Drug Administration, Bethesda, MD, USA
4Present address: Department of Immunology, Duke University Medical Center, Durham, NC
#These authors share joint senior authorship
*: Corresponding author,
Dr. Alan Sher
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
Tel: (301)-496-3535, Fax: (301)-402-0890, E-mail: [email protected]
Running Title: Requirements for CCR5 antagonism by a protozoan cyclophilin
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SUMMARY
The protozoan parasite Toxoplasma gondii possesses a protein, cyclophilin-18 (C-18),
which binds to the chemokine receptor CCR5, induces IL-12 production from murine dendritic
cells and inhibits fusion and infectivity of HIV-1 R5 viruses by co-receptor antagonism. Site
directed mutagenesis was employed to identify the domains in C-18 responsible for its CCR5
binding and anti-viral functions. To do so we focused on amino acid differences with
Plasmodium falciparum cyclophilin, which although 53% identical with C-18 has minimal
binding activity for CCR5 and generated 22 mutants with substitutions in the regions of non-
homology located on the putative surface of the molecule. Two mutations situated on the face of
C-18 predicted to be involved in its interaction with the ligand Cyclosporin A were shown to be
critical for CCR5-binding and the inhibition of HIV-1 fusion and infectivity. In contrast, four
mutations in C-18 specifically designed to abolish the peptidyl prolyl cis-trans isomerase activity
of the protein failed to inactivate its CCR5 binding and HIV inhibitory activities. IL-12 induction
by C-18, on the other hand, was abrogated by mutations effecting either the CCR5 binding or
enzymatic function of the molecule. These findings shed light on the structural basis of the
molecular mimicry of chemokine function by a pathogen derived protein and provide a basis for
further modification of C-18 into an anti-viral agent.
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INTRODUCTION
Blockade of viral infectivity is a major strategy for intervention in HIV infection (1). HIV
entry into host T lymphocyte and monocyte/macrophages has been shown to be critically
dependent on the interaction of the viral envelope with CD4 together with a chemokine co-
receptor, CCR5 or CXCR4 (2-7). Although several other chemokine receptors (CCR2, 3, 8, BOB
and others) can promote infection in vitro by specific HIV-1 variants (8), their role in vivo is
limited (9). HIV-1 isolates which interact with CCR5 (R5 type) initiate most infections and
individuals with a genetic deletion (∆32) in the CCR5 open frame appear to be highly protected
from HIV disease (10,11). These observations suggest that agents which block R5 type HIV-
CCR5 interaction may be particularly effective in preventing HIV-1 infection. Accordingly, a
series of chemokine based CCR5 antagonists have been developed and tested with partial
success in clinical trials (12).
A number of pathogens have evolved molecules that can function as mimics of
chemokines or chemokine receptors (13,14). We have recently found that the protozoan parasite
Toxoplasma gondii possesses a protein (C-18) that binds to the CC chemokine receptor CCR5,
triggers CCR5 dependent chemotaxis and induces the production of Interleukin-12 (IL-12) by
murine dendritic cells. In subsequent studies, C-18 was shown to inhibit both syncytia formation
and infectivity of R5 but not X4 HIV viruses for human T cells (15,16). Sequence analysis
revealed C-18 to be an isoform of T.gondii cyclophilin (17). Since cyclophilins from mammalian
species and from a closely related apicomplexan protozoan parasite, Plasmodium falciparum
failed to bind significantly to CCR5, induce IL-12 or possess anti-viral activity, the T.gondii
protein appears to have acquired β-chemokine-like functions as a consequence of molecular
mimicry (16). The structural basis of this mimicry is presently unclear since C-18 has no
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sequence homology with the host CCR5 ligands MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5
or MCP-2/CCL8. Cyclophilins are peptidyl prolyl isomerases and this enzyme activity is
inhibited by the drug Cyclosporin A (CsA). Interestingly, CsA was found to block the anti-viral
and IL-12 inducing functions of C-18 suggesting that either the isomerase activity of the
molecule or a structural motif conformationally altered by CsA ligation is required for C-18-
chemokine receptor interaction.
C-18 has several properties that make it attractive as a candidate HIV co-receptor
antagonist. Unlike other chemokine-based antagonists, C-18 blocks HIV interaction with T cells
and macrophages with comparable efficiency (16). Moreover, the protein does not appear to
induce significant CCR5 internalization, a potential disadvantage of a number of existing
antagonists. Finally, recent experiments (unpublished observations) indicate that C-18 also binds
to rhesus CCR5 and blocks fusion with simian immunodeficiency viral envelope, thus allowing
pre-clinical evaluation of its anti-viral activity in an in vivo primate model. Nevertheless, because
of its modest activity in both HIV fusion and infectivity assays and probable immunogenicity, it
is likely that the protein would need to be structurally optimized before use as a clinical agent. In
order to do so, an understanding of the requirements for the interaction of C-18 with CCR5 is
necessary. We have approached this problem in the present study by site directed mutagenesis of
the C-18 protein. Our findings identify a region in the C-18 molecule involved in both CCR5
binding and viral inhibition and formally establish that its peptidyl prolyl isomerase activity is
not required for either biological function. At the same time our data shed light on the molecular
basis of host chemokine mimicry by this protozoan protein.
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EXPERIMENTAL PROCEDURES
Reagents and Experimental Animals―CsA, α-Chymotrypsin (Type I-S from bovine pancreases),
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were purchased from Sigma (St. Louis, MO). Paired
antibodies against IL-12 were obtained from BD Pharmingen (San Diego, CA) for measurement
of this cytokine by ELISA. C57BL/6 (WT) and CCR5-deficient mice were obtained from the
Jackson Laboratories (Bar Harbor, ME). TLR4-deficient mice were generously provided by Drs.
S. Akira (Osaka University, Japan) and D. Golenbock (University of Massachusetts, Worcester,
MA). All animals were maintained at the an American Association of Laboratory Animal Care-
accredited National Institute of Allergy and Infectious Diseases animal facility and 8-12 weeks
old female mice were employed in all experiments.
Site-directed mutagenesis of candidate CCR5 binding determinants in C-18―The structure of
C-18 was modeled based on the crystal structure of P. falciparum cyclophilin with
Cyclosporin A bound (18) (PCyp19). Substitution of PCyp19 amino acid coordinates with a set
representing the C-18 sequence were made using alignments generated with the automated
Swiss-Model server. The initial model was energy-minimized by 200 cycles of steepest descents,
followed by 200 cycles of conjugate gradient minimization using Discover. The quality of model
geometry was checked using a Ramachandran plot. Coordinates for CsA were derived from the
PCyp19 model and added to the C-18 model after superposition of the C-18 and PCyp19
polypeptide chains using Insight II. The goal of the modeling was not to predict specific binding
interactions but rather to identify potential solvent-exposed amino acid side chains for site-
directed mutagenesis experiments. Only putatively exposed amino acids which are not conserved
between T. gondii and P. falciparum cyclophilins, C-18 and PCyp19 respectively, were selected
for the mutagenesis screen.
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The generation of the C-18 expression construct in pCRT7/CT-TOPO vector (Invitrogen, NY)
has been described previously (15). Site-directed mutagenesis of C-18 within this vector was
performed using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The following
primers were used to generate C-18 mutants:
Y10A 5´-CAGAAAGGCGGCTATGGATATCGAC-3´,
GEH17-19AAA 5´-CGACATCGACGCAGCAGCTGCCGGGCGC-3´,
E29A 5´-CTTGGAGCTCCGTGCTGACATCGCTCC-3´,
FDK43-45AAA 5´-CTTCATTGGCCTTGCTGCTGCTTACAAGGGCAGCG-3´,
D57A 5´-GTATCATCCCCGCTTTCATGATCCAG-3´,
FE65-66AA 5´-CCAGGGAGGAGATGCTGCTAACCACAACGGCAC-3´,
H68A 5´-GATTTCGAGAACGCTAACGGCACTG-3´,
H74A 5´-CACTGGAGGAGCTAGCATCTACGGC-3´,
R80A 5´-CTACGGCCGAGCTTTTGACGACGAAAAC-3´,
D82A 5´-CGGCCGAAGATTTGCTGACGAAAACTTTG-3´,
DL87-88AA 5´-GACGAAAACTTTGCTGCTAAGCACGAGCGAG-3´,
R92A 5´-GATTTGAAGCACGAGGCTGGCGTCATCTC-3´,
KTE115-117AAA 5´-CATCACCACCGTGGCTGCTGCTTGGCTCGACGCC-3´,
R122A 5´-GGCTCGACGCCGCTCACGTTGTTTTCG-3´,
IT129-130AA 5´-GTTTTCGGGAAGGCTGCAACTGAGTCGTG-3´,
SWP133-135AAA 5´-GATCACAACTGAGGCTGCTGCTACCGTCCAGGC-3´,
Q138A 5´-GTGGCCTACCGTCGCTGCTATTGAGGCTCTC-3´,
L143A 5´-CTATTGAGGCTGCTGGCGGCAGCGGCGGC-3´,
S146A 5´-GAGGCTCTCGGCGGCGCTGGCGGCCGCCCGTC-3´,
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RP149-150YV 5´-GGCAGCGGCGGCTACGTGTCTAAGGTCGCGAAAATC-3´,
S151A 5´-GCGGCCGCCCGGCTAAGGTCGCG-3´,
K155A 5´-GCTAAGGTCGCGGCTATCACGGACATTGG-3´.
The constructs in pCRT7/CT-TOPO vector (Invitrogen, Grand Island, NY) were sequenced
using vector-based external primers to confirm the introduction of the mutations.
Expression and purification of recombinant C-18 wild-type and mutant proteins―Plasmids
encoding C-18 or its mutants were transformed into Escherichia coli, BL21(DE3)pLys
(Invitrogen, Grand Island, NY). Purified inclusion bodies obtained from bacteria induced with
isopropyl-1-thio-β-D-galactopyranoside (Invitrogen, Grand Island, NY) for 4 h were next
solubilized in 6M guanidine-HCl. The solution was then added dropwise to a refolding buffer
(20 mM Tris-HCl, pH 8.0, 150 mM NaCl), followed by dialysis against 10 mM Tris-HCl, pH 8.0.
Finally, the dialyzed samples were concentrated by ultrafiltration and the recombinant protein
purified by anion-exchange chromatography on HiPrep 16/10 Q XL column (Amersham
Biosciences, Piscataway, NJ) using 10 mM Tris-HCl, pH 8.0, 1 M NaCl for elution.
CCR5 binding assay―Both competitive and direct binding assays were used to quantitate the
interaction of the C-18 proteins to cell bound CCR5. Recombinant C-18 and its mutants were
trace labeled with 125I by Phoenix Pharmaceuticals (San Carlos, CA). CEM (American Type
Culture Collection, Manassas, VA; no. CCL-119, CCR5-) cells and CEM.NKR.CCR5 (19) (a
human CCR5 transfectant of the same parental line) were incubated in triplicate in 96 well plates
(Wallac, Turku, Finland) at 105 /well with 125I-labeled C-18 alone or in the presence of human
MIP-1β (Biosource, Camarillo, CA), C-18 mutant proteins or CsA for 90 min at 4 °C (indirect
binding assay). To assess the direct binding of the C-18 mutants, CCR5+ and CCR5- cells were
incubated with increasing concentrations of 125I-labeled D82A, RP149-150YV and K155A
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mutants. Cells were then washed and the bound fraction measured radioactively as described
earlier (15). After subtraction of the non-specific binding to CCR5- cells, the molar amount of
CCR5 bound C-18 per well was calculated.
Recombinant vaccinia viruses and fusion inhibition assay―Recombinant vaccinia viruses
vCB28 (JR-FL envelope), vCB43 (Ba-L envelope), and vCB39 (ADA envelope) were kindly
provided by Christopher Broder (Uniformed Services University of the Health Sciences,
Bethesda, MD) (20). Syncytium formation was measured at different times after coculture (1:1
ratio, 1 x 105 cells each, in triplicate) of target cells (expressing CD4 and coreceptors) and
effectors (CD4–12E1 cells (21) infected overnight with 10 pfu/cell of recombinant vaccinia
viruses expressing HIV-1 envelopes). Serially diluted inhibitors were added to the target cells for
60 minutes at 37°C in a humidified CO2 incubator (3 wells per group). Effector cells were added,
and syncytium formation was followed for 3 to 4 hours. Linear regression curves were generated
and used to calculate the 50% inhibitory dose (ID50).
Viral infectivity assay―The R5 viruses BaL and JR-CSF were obtained from the NIH AIDS
Research and Reference Reagent Program (McKesson BioServices, Rockville, MD). Viral stocks
were produced and titered in phytohemagglutinin (PHA)–activated peripheral blood mononuclear
cells (PBMCs). For viral neutralization serially diluted C-18 and the C-18 mutants were added to
PM1 target cells in 96-well plates (5 x 104 cells/well, 5 replicates per group). After 90 minutes of
incubation at 37°C in a CO2 incubator, virus was added (50 tissue culture infectious dose
[TCID50]/well). After 24 hours of incubation at 37°C, unbound virus and inhibitors were washed
away, and the plates were cultured for 2 weeks. Supernatants were removed every second day,
and the cultures were supplemented with fresh medium. Viral production was determined by
measuring p24 in culture supernatants using a commercial enzyme-linked immunosorbent assay
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kit (NEN Life Sciences Products, Boston, MA). p24 production was measured every second day
for 2 weeks, and the ID50 values were based on results obtained near peak virus production
(usually between days 5-7). Viral neutralization is expressed as 50% inhibitory dose (ID50).
Generation of C-18 mutants lacking peptidyl prolyl isomerase activity―Amino acids residues
forming the substrate binding cleft are highly conserved among cyclophilins from different
species (17,22,23). We selected four of these (R53, F58, W118 and H123) in the predicted CsA-
binding pocket of C-18 for the inactivation of the peptidyl prolyl isomerase activity by site-
directed mutagenesis as described above using the following primers:
R53A 5´-GTTTTCCACGCTATCATCCCCGAC-3´,
F58A 5´-CATCCCCGACGCCATGATCCAGG-3´,
W118A 5´-CGTGAAGACAGAGGCGCTCGACGC-3´,
H123Q 5´-CGACGCCAGACAAGTTGTTTTC-3´.
The prolyl cis-trans isomerase activity of the recombinant proteins was evaluated by a
photometric assay based on the isomerization of the N-succinyl-AAPF p-nitroanilide (24,15). In
brief, isomerization of the substrate N-succinyl-AAPF p-nitroanilide (dissolved in DMSO, 10
mM stock) by C-18 or its mutants (8nM each) was determined by hydrolysis of trans prolyl
product with chymotrypsin at 5° C in 40 mM Tris HCl, pH 8.0, 100 mM NaCl followed by
measurement of p-nitroaniline absorbance at 390 nm. Enzymatic activity was assayed at
substrate concentration 100 uM. Absorbance changes over the course of hydrolysis were fit to a
single exponential function using Sigma Plot, and values for the observed first-order rate
constant (ko) were compared for wild-type and mutant proteins.
Measurement of IL-12 inducing activity of C-18 proteins―Splenic DCs were partially purified
from spleen of C57BL/6 mice as previously described (15). For measurement of IL-12
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production DCs were resuspended in cell culture medium at 106 cells/ml and distributed in 96
well plates. Recombinant C-18 and mutant proteins were then added at 10 ug/ml and the cultures
stimulated overnight. IL-12p40 levels were measured by ELISA (15). To rule out the effects of
LPS contamination, splenic DCs from CCR5-/- deficient mice, which cannot be activated by C-
18 and from TLR4 knockout mice, which are fully responsive to C-18, but LPS non-responsive
were used as controls.
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RESULTS
Selection of sites for mutagenesis based on structural comparison of C-18 with P. falciparum
cyclophilin―We have previously shown that T. gondii, but not P. falciparum or human
cyclophilins display CCR5 binding and HIV-1 anti-viral activity (16). This observation provided
a strategy for the identification of the unique structural elements in C-18 that determine its
chemokine-like activity. We first developed a model of the entire C-18 molecule based on
homology with the known crystal structure of P. falciparum cyclophilin (PfCyp19) together with
its CsA ligand (Fig. 1A) (18). We next identified those amino acids located on the putative
molecular surface of C-18 that represent non-conservative substitutions of the corresponding
amino acids in PfCyp19 (Fig. 1A and 1B). Site directed mutagenesis was then performed, in each
case substituting an Ala for the C-18 residue or consecutive residues targeted. An exception was
mutant RP149-150 in which a double substitution was made to Tyr-Val, the corresponding amino
acids present in PfCyp19 (Fig. 1B). All of the mutant proteins were successfully expressed in
E.coli although mutant R92 could not be refolded in soluble form (data not shown) and thus was
eliminated from further analysis.
Specific mutations in C-18 reduce its CCR5 binding activity―Competition of I 125 labeled C-18
interaction with CCR5 transfected cells was used to screen the mutants for altered chemokine
receptor binding. This assay revealed significantly reduced competition by three (GEH17-
19AAA, D82A and RP149-150YV) of the 21 mutants tested (Fig. 2). To confirm the validity of
the competition assay, direct binding assays were performed using representative mutants. As
shown in Fig. 3, iodinated D82A and RP149-150YV each showed greatly reduced binding to
CCR5 transfected cells in comparison to iodinated C-18 or K155A, a control mutant showing no
loss in activity in the initial screen. Interestingly, two (GEH17-19AAA and RP149-150YV) of
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the 3 loss-in-function mutations identified in Fig. 2 are situated on adjacent loops in the C-18
model, with RP149-150YV bordering the CsA binding pocket (Fig. 1), implicating this region of
the protein in CCR5 interaction. Indeed, as observed previously in other assays of C-18 function
(15,16), the addition of CsA dramatically inhibited the binding of labeled C-18 to the indicator
cells (Fig. 3).
C-18 mutants with decreased CCR5 binding also display reduced anti-viral activity―The same
mutant proteins that were assessed for CCR5 interaction were also screened for their ability to
inhibit HIV-1 envelope dependent cell fusion in an assay employing CD4/CCR5- expressing
PM1 target cells. Using 12E1 effector cells expressing HIV-1 R5 envelope (JR-FL), wild–type
C-18 blocked fusion with ID50 values ranging between 3-10 µg/ml in 5 experiments. The same
three mutants described above as showing impaired CCR5 binding showed decreased viral
fusion inhibition with very significant increases in the ID50 values (Fig. 4A+B). The reduced
anti-viral activity of proteins GEH17-19AAA, D82A and RP149-150YV was confirmed in a
separate set of fusion assay employing a different R5 viral envelope (Bal). The findings in the
fusion assays were reproduced in virus infectivity assays in which the ability of the proteins to
block entry into human PBMC was compared (Fig. 5). Again mutants GEH17-19AAA and
RP149-150YV showed reduced activity. However, in this assay system we were unsuccessful in
generating an ID50 value for D82A even using excessively high concentrations of the mutant
protein.
The peptidyl-prolyl isomerase enzymatic activity of C-18 is not required for CCR5 binding or
anti-viral activity―As noted previously, the C-18 ligand CsA inhibits both the anti-viral (16)
and CCR5 binding (Fig. 3) activities of the protein. Since CsA is a potent antagonist of the
peptidyl prolyl isomerase activity of cyclophilins, it was possible that this enzymatic function is
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required for the biological properties of T.gondii C-18. To test this hypothesis we generated four
additional mutant proteins within the highly conserved substrate-binding pocket of C-18. These
mutations had previously been shown to abrogate enzyme activity in human cyclophilin (25) and
this loss in activity was confirmed in the C-18 mutants (Fig. 6A and Supplementary Table 1).
Importantly, none of the four mutant proteins lost CCR5 binding (Fig. 6B) or anti-viral activity
as measured in the fusion and infectivity assays (Fig. 6C+D). These data formally demonstrate
that the CCR5 dependent biological functions of C-18 do not depend on its enzymatic activity.
In the same series of experiments, we showed that the original series of C-18 mutant
proteins studied above (Fig. 1-5) retain their enzymatic activity (Fig. 6A and Supplemental
Table 1). The latter data argues that these recombinant molecules were properly folded following
expression and purification. An exception however, was mutant D82A which while displaying
loss in fusion inhibition and CCR5 binding also lacked enzymatic activity (data not shown) and
as described above was unexpectedly totally lacking in activity in the inhibition of HIV
infectivity assay. Based on this combined evidence we concluded that the loss in biological
function observed with this mutant protein is likely to be the result of improper folding.
IL-12 induction by C-18 requires both the CCR5 binding and enzymatic structural domains of
C-18 ―C-18 was originally described as a parasite derived inducer of IL-12 (15,26), a cytokine
involved in host resistance to T.gondii infection (27). To confirm the role of CCR5 binding in
this activity, wild-type and mutant C-18 proteins were tested for their ability to induce IL-12
from cultures of murine splenic dendritic cells. As shown in Fig. 7 the same mutants (GEH17-
19AAA and RP149-150YV) which showed loss in CCR5 binding and anti-viral function also
showed impaired IL-12 inducing activity. Interestingly, however, the proteins with mutated
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peptidyl prolyl isomerase enzymatic activity also showed decreased IL-12 induction despite their
normal CCR5 binding.
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DISCUSSION
We have previously demonstrated that T.gondii C-18 evolved the ability to bind both
mouse and human CCR5 and to trigger chemokine-like functions in cells bearing this receptor
(15,16). The fact that the corresponding cyclophilin (PfCyp19) from P.falciparum, a closely
related apicomplexan protozoan, lacks significant receptor binding activity suggested a
molecular approach for understanding the basis of the structural changes leading to CCR5
targeting. Since C-18 and PfCyP19 share a 63% amino acid homology, we hypothesized that the
critical sequence differences in C-18 responsible for its chemokine receptor interaction are
located on the exposed surface of the molecule, and identified a set of spatially distinct T.gondii
specific amino acid residues as candidate determinants of CCR5 binding activity (Fig. 1). Amino
acid differences in the internal region of the molecule were ignored in the present study but could
conceivably contribute to its proper folding and receptor binding.
Site directed mutagenesis revealed that 2 of the 22 non-homologous single amino acids or
sequences chosen for analysis were critical for CCR5 binding as well as inhibition of R5 viral
fusion and infectivity. Interestingly, these two disabling mutations are located in the N and C-
termini of the protein. However, in the predicted 3 dimensional structure of C-18, the amino
acids in question lie in close proximity to each other (within 20 Ao) on adjacent loops of a β-
sheet structure (Fig. 1). The latter observation suggests that CCR5 binding may have evolved as
a result of a limited number of mutations in regions that are separated in the primary structure of
the molecule, but adjacent in the tertiary structure. Given that CCR5 binding of conventional
chemokine ligands involves multiple extracellular receptor loops, it is not surprising that
spatially distinct structural elements on C-18 would be required for receptor interaction. Indeed,
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in previous studies, multiple and spatially distinct amino acid substitutions were shown to
abrogate the binding of MIP-1β to human CCR5 (28,29).
CsA is a known antagonist of the peptidyl prolyl isomerase activity of cyclophilins and
we previously demonstrated that CsA blocks the anti-viral activity of C-18 and as demonstrated
here inhibits its CCR5 binding. Nevertheless, the amino acid sequences that determine the
substrate interaction are highly conserved between phylogentically distinct cyclophilins
including those with no known CCR5 binding activity (22). This observation led us to
hypothesize that the enzymatic activity of C-18 is not important for its mimicked chemokine
functions (16). To directly test this hypothesis we constructed four C-18 mutants with amino acid
substitutions in the conserved substrate pocket which led to loss in enzymatic function. These
mutations failed to diminish either the CCR5 binding or anti-viral activities of C-18. Moreover,
neither of the two mutations studied which did result in loss in chemokine receptor binding
altered the enzymatic activity of the protein. These findings formally establish that the peptidyl
prolyl isomerase activity of C-18 is not required for its anti-viral activity.
While in our experimental read-out C-18 is the only cyclophilin tested with anti-viral
activity, other groups have described HIV inhibitory functions associated with mammalian
cyclophilins. Host-derived cyclophilin A has been shown to be incorporated into HIV during
virion assembly through interaction with a proline-rich domain in the capsid protein and its
presence is known to be essential for infectivity (30-32). Incubation with excess human
cyclophilin has been shown in some (33,34) but not all (35) reports to block HIV fusion and/or
infectivity of both R5 and X4 viruses. This inhibition has been proposed to result from
competition of the binding of cyclophilin on the viral particle with Heparan and CD147 on the
target cell membrane leading to decreased virion attachment (34,36). The anti-viral activity of
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C-18 does not appear to involve the same mechanism since its effects are restricted to R5 viruses
and due to specific interaction with CCR5, a property not shared by human and other
cyclophilins. Moreover, the interaction of human cyclophilin with host cell CD147 has been
shown to require the peptidyl prolyl isomerase activity of the former molecule (37) and as
demonstrated here enzymatic function is not necessary for the anti-viral properties of C-18.
Why did T.gondii evolve a cyclophilin molecule with the ability to bind to CCR5? While
the explanation for this apparent molecular mimicry is not clear, our previous studies suggest
that this property may relate to the host-parasite interplay in toxoplasma infection. T.gondii
induces a potent cell-mediated immune response early in infection which prevents the parasite
from overwhelming its intermediate hosts and drives it into a latent state necessary for
transmission. Previous studies have indicated that the production of IL-12 by tachyzoite
stimulated dendritic cells (DCs) is a critical element in this control of acute infection and that
CCR5 is an important element in this response (26, 27). Importantly, C-18 was shown to
stimulate IL-12 production by DCs (although not as potently as the parasite extract from which it
was purified) and to do so in a CCR5 dependent manner. On the basis of this evidence we
speculated that C-18 developed the capacity to act as a CCR5 ligand in order to promote IL-12
mediated host control of the parasite. Indeed, the same mutations in the protein that diminished
CCR5 binding also ablated its DCs IL-12 inducing activity (Fig. 6). Based on the latter
hypothesis, the anti-HIV activity of C-18 is likely to be a co-incidence reflecting the common
usage of CCR5 by two phylogenetically distinct pathogens. Whether the interaction of C-18 with
CCR5 impacts on HIV progression in individuals co-infected with this opportunistic pathogen
remains a matter for speculation.
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Regardless of its exact biological function, C-18 as a CCR5 ligand of microbial origin
maybe of therapeutic interest as co-receptor antagonist for R5 viruses. Admittedly to be used in
this manner, C-18 would have to be structurally modified to eliminate any deleterious host
interactions and if possible, its affinity for its receptor increased. Other important parameters to
be considered include pharmokinetics, bioavailability, and unwanted immunogenicity. The
present study provides an important first step in the design of such a molecule by defining a
major region in C-18 that appears necessary for receptor interaction and by clearly demonstrating
that the peptidyl prolyl isomerase activity of the protein can be deleted without any significant
loss in anti-viral function. Since many of the potential unwanted side-effects of cyclophilin
administration are likely to result from the enzyme activity of the molecule, the latter observation
is clearly of importance in reducing potential drug toxicity. In this context, it should be pointed
out that while C-18 is able to stimulate IL-12 production by murine CD8α+ DCs, we so far have
been unable to identify a responsive DCs population in humans (unpublished observations).
Therefore, the IL-12 inducing activity of C-18 observed with murine DCs may be an irrelevant
concern in designing protein for human use. Moreover, as demonstrated here mutation of the
peptidyl prolyl isomerase activity of C-18 was found to destroy its IL-12 inducing function
without impairing its CCR5 binding and anti-viral properties.
Having identified regions in C-18 that are critical for CCR5 binding, we can now
examine sequence substitutions that might enhance this activity. In this regard, it was of interest
that two of the mutations (E29A and K155A) resulted in small but reproducible increases in anti-
viral and CCR5 binding activity (Fig. 4). It is hoped that by continued mutational analysis,
sequence alterations will be revealed that will further enhance the binding of C-18 to CCR5 and
therefore lead to potentially increased efficacy of the molecule as an anti-retroviral.
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ACKNOWLEDGEMENTS
We thank Edward Berger for helpful comments and Jose Ribeiro for his advice and
encouragement of this project.
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FIGURE LEGENDS
Figure 1. Selection of amino acids in C-18 for site-directed mutagenesis based on sequence
difference with PfCyp19. A, Space-filling model of C-18-CsA complex. Amino acid residues
that were not mutated are colored red. Mutated residues that had no significant effect in
bioassays are colored yellow. CsA is labeled and colored blue. Residues representing the
mutation GEH17-19AAA are colored cyan and labeled A. Residues representing mutation
RP149-150YV are colored cyan and labeled B. B, Sequence alignment of C-18 and PfCyp19
with regions of identity high-lighted in yellow. The amino acids selected for site-directed
mutagenesis (Ala substitution) are indicated with an arrow. An exception was mutant RP149-150
in which a double substitution was made to Tyr- Val, the corresponding amino acids present in
PfCyp19.
Figure 2. Site directed mutagenesis of C-18 results in selective loss of CCR5 binding. 125I-
labeled C-18 at 1 nM concentration was incubated with CCR5+ and CCR5- cells alone or in the
presence of a 10:1 ( A) or 100:1 excess (B) of cold human MIP-1β, wild –type C-18 or mutant
proteins for 90 min at 4 °C. Cells were then washed and the bound fraction measured
radioactively. Specific binding was calculated by subtracting the non-specific background
observed with CCR5- cells. The data shown are means of 4 experiments ± SD.
Figure 3. Affinity of C-18 vs C-18 mutants for CCR5 as determined by direct binding. 125I-
labeled C-18, K155A, RP149-150YV and D82A mutant proteins were incubated with CCR5+
and CCR5- cells for 90 min at 4 °C. Cells were then washed and the bound fraction measured
radioactively. In one reaction, 125I-labeled C-18 was preincubated with 1 uM of CsA for 1 h at
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37C before addition of the CCR5 expressing cells. The experiment shown is representative of
three independent experiments with similar results.
Figure 4. Inhibition of HIV-1 envelope–mediated cell fusion by C-18 and its mutants. PM1
cells were incubated with serial dilutions of C-18 or mutant C-18 proteins for one hour at 37°C
and then mixed (1:1, in triplicate) with 12E1 cells infected previously with recombinant vaccinia
(vCB28) expressing JR-FL (A + B) or Bal (C) envelopes. Syncytia were scored after 3-4 hours
of incubation and ID50 values calculated. The data in (A) represent the mean ± SD of the ratio of
ID50 values of mutant to wild C-18 proteins in 4 different experiments. Representative
experiments are also shown for JR-FL (B) and Bal (C) envelopes.
Figure 5. Mutations in C-18 that abrogate CCR5 binding also result in reduced inhibition
of HIV infectivity. C-18 and the mutant proteins GEH17-19AAA and RP149-150YV were
added to PM1 target cells and after 90 minutes of incubation at 37°C virus added. After an
additional 24 hrs unbound virus and inhibitors were washed away, and the plates incubated for 2
weeks. Viral production was then assessed by measuring p24 in culture supernatants. ID50 values
were based on results obtained near peak virus production (between days 5-7). The experiment
shown is representative of two performed.
Figure 6. The peptidyl-prolyl isomerase activity of C-18 is not required for its interaction
with CCR5. A, Peptidyl prolyl isomerase activity of C-18 and representative mutants as
expressed by the generation of p-nitroanilide measured at A390 from its substrate. The loss in
enzyme activity of two mutants R53A and W118A is shown. An identical loss was observed
with mutants F58A, H132Q and D82A (not shown). The calculated Ko values for the remaining
proteins are presented in Supplemental Table 1 as evidence of proper refolding. B, The retention
of CCR5 binding by the mutant proteins with inactivated enzymatic activity was confirmed
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based on their ability to compete the interaction of 125I-labeled C-18 with CCR5+ cells as
described in Fig. 2. C, Retention of anti-viral activity of C-18 proteins with mutated peptidyl
prolyl isomerase activity as evaluated by cell fusion (C) and by infectivity (D) assays using
similar protocols as described in Fig. 4 and Fig. 5.
Figure 7. IL-12 induction by C-18 requires both CCR5 binding and intact peptidyl prolyl
isomerase enzymatic activities. A, Semi-purified DCs from WT (black bars), CCR5 (white
bars) and TLR4 deficient mice (grey bars) were stimulated with C-18 or mutant proteins
(10 ug/ml) and IL-12p40 was measured in supernatants 20h later. B, In a separate experiment
WT splenic DCs were incubated with C-18 proteins in which the enzymatic activity was
impaired by mutation. Bars represent means ± SD of duplicate ELISA values. Similar results
were obtained in three repeat experiments. The CCR5 and TLR4 deficient DCs were used as
controls for CCR5 involvement and LPS contamination respectively.
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FOOTNOTES
The abbreviations used are: C-18, cyclophilin-18; PCyp19, Plasmodium falciparum cyclophilin;
CsA, Cyclosporin A; IL-12, Interleukin-12; WT, wild type C57BL/6 animals; CCR5- cells,
(American Type Culture Collection, number CCL-119); CCR5+ cells, a human CCR5
transfectant of the CCL-119 cell line; DCs, dendritic cells; ID50, 50% inhibitory dose.
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Supplementary Table 1. Peptidyl prolyl isomerase activity of C-18 and its mutants. The
enzymatic activity of the recombinant proteins was evaluated by a photometric assay based on
the isomerization of the N-succinyl-AAPF p-nitroanilide. The data were fit to a single
exponential and the rate constants were used to estimate steady state kinetic parameter k0.
Protein ko, s-1 SD, s-1
C-18 0.02885 0.000235
Y10A 0.02995 0.000495
GEH17-19AAA 0.0193 0.003253
E29A 0.02095 0.002192
FDK43-45AAA 0.01615 0.0007
D57A 0.027 0.00099
FE65-66AA 0.0166 0.000849
H68A 0.02115 0.00005
H74A 0.0164 0.000141
R80A 0.0265 0.000141
DL87-88AA 0.0168 0.001414
KTE115-117AAA 0.021 0.000707
R122A 0.0162 0.000707
IT129-30AA 0.0164 0.000495
SWP133-135AAA 0.017 0.000283
Q138A 0.0205 0.000283
L143A 0.01745 0.001485
S146A 0.01735 0.000636
S151A 0.021 0.000707
K155A 0.027 0.000849
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Figure 1.
A.
T. gondii MENAGVRKAYMDIDIDGEHAGRIILELREDIAPKTVKNFIGLFD-----------KYKGS
P. falcip --MSKRSKVFFDISIDNSNAGRIIFELFSDITPRTCENFRALCTGEKIGSRGKNLHYKNS
T. gondii VFHRIIPDFMIQGGDFENHNGTGGHSIYGRRFDDENFDLKHER-GVISMANAGPNTNGSQ
P. falcip IFHRIIPQFMCQGGDITNGNGSGGESIYGRSFTDENFNMKHDQPGLLSMANAGPNTNSSQ
T. gondii FFITTVKTEWLDARHVVFGKITTESWPTVQAIEALGGSGGRPSKVAKITDIGLLE
P. falcip FFITLVPCPWLDGKHVVFGKVI-EGMNVVREMEKEGAKSGYVKRSVVITDCGEL-
1
50
10 17-19 29 4344
57 6566 68 74 80 82 8788 92
115-117 122 129130133-135 138 143 146 149 155
109
45B.
151
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Figure 2.
0
100
200
300
400
500
600
700
800
900
1000
Boun
d C
-18
(fmol
e)
A.
Competitor
KTE
115-
117
SW
P13
3-13
5
MIP
-1β
C-1
8
Y10A
GE
H17
-19
E29
A
FDK
43-4
5D
57A
FE65
-66A
A
H68
A
H74
A
R80
A
D82
A
DL8
7-88
AA
R12
2A
IT12
9-13
0AA
Q13
8A
L143
A
S14
6A
RP
149-
150Y
V
S15
1AK
155A
-
C-18 mutant proteins
0
100
200
300
400
500
600
700
800
Boun
d C
-18
(fmol
e)
B.
Competitor
KTE
115-
117
SW
P13
3-13
5
MIP
-1β
C-1
8
Y10A
GE
H17
-19
E29
A
FDK
43-4
5D
57A
FE65
-66A
A
H68
A
H74
A
R80
A
D82
A
DL8
7-88
AA
R12
2A
IT12
9-13
0AA
Q13
8A
L143
A
S14
6A
RP
149-
150Y
V
S15
1AK
155A
-
C-18 mutant proteins
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Figure 3.
0
2000
4000
6000
8000
10000
1 2 3 4 50 0.3 1 3 9
Bou
nd L
igan
d (fm
ole)
C-18
K155ARP149-150YVD82A
C-18 + CsA
Ligand, (nM)
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Figure 4.
0
20
40
60
80
100
0
2
4
6
8
10
12
14
16
wt C
18
Y10A
GEH
17AA
A
E29A
FDK4
2AAA
D56
A
FE64
AA
H67
A
H73
A
R80
A
D82
A
DL8
7AA
KTE1
15AA
A
R12
2A
IT12
9AA
SWP1
33AA
A
Q13
8A
L143
A
S146
A
RP1
45YV
S151
A
K155
A
B.
0
0
10
20
30
40
50
wt C18 GEH17AAA D82A RP145YV
rela
tive
units
ID50
, ug/
ml
A.
4
8
12
16
C.
ID50
, ug/
ml
C-18 GEH17-19AAA D82A RP149-150YV
KTE
115-
117
SW
P13
3-13
5
C-1
8
Y10A
GE
H17
-19
E29
A
FDK
43-4
5
D57
A
FE65
-66A
A
H68
A
H74
A
R80
A
D82
A
DL8
7-88
AA
R12
2A
IT12
9-13
0AA
Q13
8A
L143
A
S14
6A
RP
149-
150Y
V
S15
1A
K15
5A
KTE
115-
117
SW
P13
3-13
5
C-1
8
Y10A
GE
H17
-19
E29
A
FDK
43-4
5
D57
A
FE65
-66A
A
H68
A
H74
A
R80
A
D82
A
DL8
7-88
AA
R12
2A
IT12
9-13
0AA
Q13
8A
L143
A
S14
6A
RP
149-
150Y
V
S15
1A
K15
5A
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Figure 5.
0
20
40
60
80
100
120ID
50, u
g/m
l
C-18 GEH17-19AAA RP149-150YV
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Figure 6.
0
200
400
600
800
- wt C-18 R53A F58A W118A H123Q
Boun
d C
-18
(fmol
e)
Competitor
A. B.
Time (sec)
A39
0
0 20 40 60 80 1001.00
1.02
1.04
1.06
1.08
1.10
1.12
1.14
1.16
1.18
C-18GEH17-19A
K155ARP149-150YV
W118AR53A
0
2
4
6
8
10
wtC-18 R53A F58A W118A H123Q
C.
0
1
2
3
4
wt C-18 R53A F58A W118A H123Q
ID50
, ug/
ml
D.ID
50, u
g/m
l
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Figure 7.
0
0.4
0.8
1.2
media wt C-18 R53A F58A W118A H123Q
IL-1
2p40
(ng/
ml)
IL-1
2p40
(ng/
ml)
B.
A.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
GEH
17-1
9
FDK
43-4
5
FE65
-66A
A
DL8
7-88
AA
IT12
9-13
0AA
RP1
49-1
50YV
C-1
8
Y10A
E29A
D57
A
H68
A
H74
A
R80
A
R12
2A
Q13
8A
L143
A
S146
A
S151
A
K15
5A
KTE
115-
117
SWP1
33-1
35
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Golding and Alan SherFelix Yarovinsky, John F. Andersen, Lisa R. King, Patricia Caspar, Julio Aliberti, Hana
toxoplasma gondiiStructural determinants of the anti-HIV activity of a CCR5 antagonist derived from
published online October 6, 2004J. Biol. Chem.
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