structural determinants of the anti-hiv activity of a ccr5 antagonist

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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