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Originally published In Press as doi:10.1074/jbc.M500236200 on June 23, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29570-29577, August 19, 2005
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CCR5 N-terminal Region Plays a Critical Role in HIV-1 Inhibition by Toxoplasma gondii-derived Cyclophilin-18*

Hana Golding{ddagger}§, Surender Khurana{ddagger}, Felix Yarovinsky¶, Lisa R. King{ddagger}, Galina Abdoulaeva||, Liselotte Antonsson**{ddagger}{ddagger}, Christer Owman**, Emily J. Platt§§, David Kabat§§, John F. Andersen¶¶, and Alan Sher¶

From the {ddagger}Division of Viral Products and ||Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, Laboratory of Parasitic Diseases and ¶¶Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892, **Division of Molecular Neurobiology, BMC-A12, SE-221 84 Lund, Sweden, and §§Oregon Health and Sciences University, Portland, Oregon 97239

Received for publication, January 7, 2005 , and in revised form, June 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular mimicry of chemokine ligands has been described for several pathogens. Toxoplasma gondii produces a protein, cyclophilin-18 (C-18), which binds to the human immunodeficiency virus (HIV) co-receptor CCR5 and inhibits fusion and infection of T cells and macrophages by R5 viruses but not by X4 viruses. We recently identified structural determinants of C-18 required for anti-HIV activity (Yarovinsky, F., Andersen, J. F., King, L. R., Caspar, P., Aliberti, J., Golding, H., and Sher, A. (2004) J. Biol. Chem. 279, 53635–53642). Here we have elucidated the fine specificity of CCR5 residues involved in binding and HIV inhibitory potential of C-18. To delineate the regions of CCR5 involved in C-18 binding, we analyzed C-18 inhibition of cells expressing CXCR4/CCR5 chimeric receptors and CCR5 with a truncated N terminus ({Delta}2–19). These experiments identified a critical role for the N terminus of CCR5 in C-18 binding and anti-HIV activity. Studies with a large panel of CCR5 N-terminal peptides, including Tyr-sulfated analogues, truncated peptides, and alanine-scanning mutants, suggested that each of the 12–17 amino acids in the N terminus of CCR5 are essential for C-18 binding and inhibitory activity. Tyr sulfation did not improve C-18 reactivity. This finding is of interest because the same CCR5 N-terminal region was shown previously to play a key role in binding of HIV-1 envelope glycoproteins. The elucidation of the functional C-18-binding mechanism may help in the rational design of novel antiviral agents against HIV.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The HIV-11 co-receptor CCR5 has been identified as a major target for HIV-1 entry inhibitors, because almost all the viruses responsible for establishing chronic infection in recipients have been typed as CCR5-tropic (R5) strains. The naturally occurring {Delta}32ccr5 allele (1, 2) when homozygous is associated with resistance to in vitro infection of CD4+ cells with R5 viruses (3, 4). Moreover, {Delta}32ccr5 homozygosity confers considerable protection against HIV infection in vivo (5, 6). Yet this genotype is not associated with abnormal immune function and may be dispensable because of redundancy in chemokine receptor usage (7). Extensive work by multiple laboratories identified the N terminus and the second extracellular loop (ECL-2) of the CCR5 and CXCR4 co-receptors as the main sites for interactions with the HIV-1 GP120 envelopes following binding to CD4 (reviewed in Zaitseva et al. (8)). The N terminus of both CCR5 and CXCR4 are rich in tyrosine residues that are often modified by sulfation in different cells (9). The sulfated tyrosines were shown to contribute to the binding of CCR5 to MIP1-{alpha}, MIP1-{beta}, and HIV-1 GP120-CD4 complexes. In addition, tyrosine-sulfated peptides based on the N terminus of CCR5 interacted with CD4-bound GP120 and could inhibit viral entry (10, 11).

Currently, there are three main classes of CCR5-targeting inhibitors under development: CC-chemokine analogues, small molecules, and monoclonal antibodies (8, 12). Several of these inhibitors have entered clinical trials (12, 13).

Our group has identified a new CCR5 agonist (C-18) derived from the protozoan parasite Toxoplasma gondii. This protein was identified as an isoform of T. gondii cyclophilin and was shown to signal via murine and human CCR5 and to induce interleukin-12 secretion from mouse dendritic cells (14, 15). In addition, T. gondii cyclophilin C-18, but not a closely related cyclophilin from the apicomplexan protozoan parasite Plasmodium falciparum, bound specifically to human CCR5 and blocked fusion and infection with multiple R5 HIV-1 isolates (16). In a recent study, we established that the peptidyl prolyl cis-trans-isomerase enzymatic activity of T. gondii cyclophilin is not required for its HIV-1 blocking activity in human cells. Furthermore, we identified several amino acids in C-18 critically required for interactions with CCR5 (17). The current study was aimed at identifying regions in CCR5 that are involved in C-18 binding. This work has identified a critical sequence in the N terminus of CCR5 that is required for C-18 interaction and anti-HIV activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Recombinant C-18 Protein—Recombinant C-18 expression and purification steps were described elsewhere (15, 17). In brief, a plasmid encoding C-18 was transformed into Escherichia coli BL21(DE3)pLys (Invitrogen). Purified inclusion bodies obtained from bacteria induced with isopropyl-1-thio-{beta}-D-galactopyranoside (Invitrogen) for 4 h were solubilized in 6 M guanidine HCl. The protein was refolded in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl buffer, dialyzed against 10 mM Tris-HCl, pH 8.0, buffer and purified on HiPrep 16/10 Q FF column (Amersham Biosciences), using 10 mM Tris-HCl, pH 8.0, 1 M NaCl for elution.



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  		FIG. 1.
Surface expression of chimeric CXCR4/CCR5 co-receptors by transfected U87.D4 cells. Stably transfected cells were stained with either mAb 2D7 (top left panel) specific for CCR5 ECL-2 or with mAb 5.5B5 (all other panels) recognizing the CXCR4 N-(2–16) region. Broken lines represent staining with isotype-matched antibody control.

 
Peptides—Table I lists the sequences of the key peptides used in the study. All peptides were synthesized at the Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, on an Applied Biosystems peptide synthesizer model 433 (Foster City, CA) by standard Fmoc (9-fluorenyl methoxycarbonyl) chemistry. After cleavage followed by precipitation and washing with methyl tert-butyl ether, peptides were purified by reversed-phase high performance liquid chromatography using Walters DeltaPak C-18 column and a linear gradient of 0.1% trifluoroacetic acid/water and 0.1% trifluoroacetic acid/acetonitrile. Fractions containing the desired peptide products were evaporated to remove acetonitrile prior to lyophilization for long term storage. For peptides with sulfotyrosine, sulfotyrosine was incorporated into the growing peptide chain, on the instrument during synthesis, by using N-a-Fmoc-O-sulfo-L-tyrosine sodium salt hydrate as the building block. The sulfotyrosine amino acid, as well as all unmodified amino acids used for this project, was purchased from Anaspec Inc. (San Jose, CA). The purified peptides were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.


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  		TABLE I
CCR5-derived peptides used in the current study

 
Peptide ELISA—Peptides/proteins were biotinylated using maleimide PEO2-biotin reagent as per the manufacturer's instructions (Pierce). The purified C-18 protein (200 ng/well) was coated onto polystyrene wells. After blocking with Eagle's modified minimal essential medium containing 5% FBS, serial dilutions (100 µl/well) of biotinylated R5-(1–18)-peptide (starting concentration of 100 nM) diluted in blocking solution were added to each well, incubated for 1 h at room temperature, followed by addition of peroxidase conjugated streptavidin. The reaction was quantified using 2,2'-azinobis-(3-ethylbenzthiazoline sulfonic acid) substrate. Absorbance was measured at 405 nm.

Competition ELISA Using Synthetic Peptides—Competition of peptides with biotinylated R5-(1–18)-peptide for binding to C-18 was performed by peptide ELISA. Fifty microliters (10 µM or 10-fold serial dilutions) of synthetic R5-(1–18)-peptide (or its mutant/shorter derivatives) or a control peptide (CXCR4, N terminus) were added to C-18-coated wells for 30 min at room temperature. Next, the biotinylated R5-(1–18)-peptide (100 nM/50 µl/well) was added for 1 h at room temperature. The reaction was developed as described for peptide ELISA.

CCR5 Binding Assay—Recombinant C-18 was trace-labeled with 125I from Phoenix Pharmaceuticals (San Carlos, CA). CEM cells (catalogue number CCL-119, CCR5–, American Type Culture Collection, Manassas, VA) and CEM.NKR.CCR5 cells (a human CCR5 transfectant of the same parental line, kindly provided by John Moore) 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 N-terminal peptides for 90 min at 21 °C (indirect binding assay). Cells were then washed, and the bound fraction measured radioactively as described earlier (17). After subtraction of the nonspecific binding to CCR5– cells, the molar amount of CCR5-bound C-18 per well was calculated. In a second binding assay, recombinant FITC-labeled C-18 was incubated with CEM and CEM.NKR.CCR5 cells for 30 min at 4 °C either alone or in the presence of the N-terminal 31-amino acid peptide. Cells were then washed, and the bound C-18 was analyzed by flow cytometry.

Recombinant Vaccinia Viruses and Fusion Inhibition Assay—Recombinant vaccinia viruses vCB28 (JR-FL envelope) and vCB43 (Ba-L envelope) were kindly provided by Christopher Broder (Uniformed Services University of the Health Sciences, Bethesda) (18). Syncytium formation was measured after co-culture (1:1 ratio, 1 x 105 cells each, in triplicate) of target cells (expressing CD4 and co-receptors) and effectors (12E1 cells, which are CD4 negative (19)) infected overnight with 10 plaque-forming units/cell of recombinant vaccinia viruses expressing HIV-1 envelopes. Serially diluted C-18 was added to the target cells for 60 min at 37 °C in a humidified CO2 incubator (three wells per group). Effector cells were added, and syncytium formation was followed for 3–4 h. Linear regression curves were generated and used to calculate the 50% inhibitory dose (ID50). To test the ability of short peptides derived from CCR5 to bind to C-18 and reverse its fusion-inhibiting activity, serially diluted peptides were preincubated with C-18 in microcentrifuge tubes (1:1 ratio) at 37 °C for 60 min. The peptide/C-18 mixtures were then added to PM1 cells (20) in 96-well plates for an additional 60 min at 37 °C, after which the effector cells were added as described above. The C-18-mediated fusion inhibition (in the absence of peptides) was considered as 100% and ranged between 55 and 75% inhibition using effector cells expressing JR-FL or Ba-L envelopes.

Stably Transfected U87.CD4 Cells Expressing Chimeric CXCR4/CCR5 Receptors—The construction of plasmids expressing either wild-type CCR5 or CXCR4/CCR5 chimeric molecules (designated FC-1, FC-2, and FC-4b) and the generation of stably transfected U87.CD4 cells expressing these chimeric molecules were described previously (21). Here we used U87.CD4 expressing either WT CCR5 or FC-1 (CXCR4 Pro-42/Pro-35 CCR5), FC-2 (CXCR4 Asp-74/Ile-67 CCR5), and FC-4b (CXCR4 Ile-185/Cys-178 CCR5) cells. Surface expression of the WT or chimeric CXCR4/CCR5 receptors in the U87.CD4 transfectants was monitored by flow cytometry using either mAb 2D7 (Pharmingen), which recognizes an epitope in the ECL-2 of CCR5, or mAb 5/5B5, which recognizes the CXCR4 N-terminal peptide (amino acids 2–16), as described previously (2, 21). The four cell lines expressed comparable levels of WT CCR5 or CXCR4/CCR5 chimeric receptors (Fig. 1).

Viral Infectivity Assay—JC.6 (HeLa-CD4 cells expressing wild-type CCR5), R5d18.2 (HeLa-CD4 cells expressing CCR5 that lacks the N-terminal amino acids 2–19), and HIV-1 variant JR-CSF18.2ad D2 (adapted to grow on CCR5 ({Delta}18))-HeLa-CD4 cells were described previously (22, 40). The HeLa-CD4 derivatives were grown in low glucose DMEM supplemented with 10% FBS and penicillin/streptomycin (DMEM/FBS medium). No selection drug was added. Twenty four hours before infection, cells were plated in flat bottom 96-well plates (2 x 103 cells per well) in 200 µl of DMEM/FBS medium. Prior to infection, the medium was removed, and cells were treated for 20 min with serum-free DMEM (low glucose) containing DEAE-dextran (8 µg/ml), followed by washing with serum-free DMEM. C-18 was added to wells containing cells for 2 h at 37 °C, after which JR-CSF (18.2ad) virus was added at 0.75 x 103 focus-forming units/well. After 24 h of incubation of cells, C-18, and virus at 37 °C in a CO2 incubator, the cells were washed extensively (to remove unbound virus and inhibitor), and fresh DMEM/FBS medium was added to all wells. The plates were cultured for 2 weeks. Supernatants were removed daily, 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 kit (PerkinElmer Life Sciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
C-18-mediated Fusion Inhibition Requires the N Terminus of CCR5—In order to delineate the regions within CCR5 that contribute to C-18 binding, we made use of a panel of U87.CD4 cells expressing chimeric CXCR4/CCR5 (or WT CCR5) receptors. All four stably transfected cell lines expressed similar levels of co-receptor (Fig. 1) and were shown previously to support infection of Ba-L (21) and several primary isolates (23). In a fusion assay, all four cell lines fused with 12E1 effector cells expressing the Ba-L envelope at similar levels (Fig. 2, see legend). Fusion of cells expressing WT CCR5 was effectively blocked by C-18, by the CCR5-agonist MIP1-{beta}, and by mAb 2D7, which targets a conformationally sensitive epitope in CCR5 ECL-2 (Fig. 2). In contrast, cells expressing the FC-1, FC-2, and FC-4b chimeric CXCR4/CCR5 receptors, which have the entire N terminus plus increasing amounts of sequences from CXCR4, fused with JR-FL envelope-expressing cells but were not blocked by either C-18 or MIP1-{beta}. mAb 2D7 blocked fusion of cells expressing wild-type CCR5, FC-1, and FC-2 chimeric co-receptors. In contrast, fusion with FC-4b-expressing cells was not inhibited by mAb 2D7, most likely because the key residues 171-KE-172 at the base of CCR5 ECL-2, which are required for 2D7 binding, were replaced by CXCR4 amino acids in this chimeric receptor (21, 24, 25) (Fig. 2). These results suggested that the CCR5 N terminus is required for C-18 binding.



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  		FIG. 2.
C-18-mediated fusion inhibition requires the N terminus of CCR5. Stably transfected U87.CD4.CCR5, U87.CD4.FC-1, U87.CD4.FC-2, and U87.CD4.FC-4b were treated with C-18 (20 µg/ml), MIP1-{beta} (10 µg/ml), or mAb 2D7 (10 µg/ml) for 60 min at 37 °C, followed by the addition of 12E1 cells expressing JR-FL envelope. Syncytia were scored after 3 h. Control syncytia (no inhibitor present) were as follows: 230 ± 12, 232 ± 13, 276 ± 4, and 192 ± 15 for the four cell lines, respectively. All groups were run in triplicate, and the standard deviations were between 10 and 15% of the means. The experiment shown is representative of three performed.

 
Peptides Derived from the CCR5 N Terminus Can Reverse C-18-mediated Fusion Inhibition—We next used a panel of synthetic peptides derived from the N terminus and the extracellular loop-2 (ECL-2) of CCR5 (Table I) to investigate further the C-18 contact sites. All the peptides used in the current study had no inhibitory or enhancing effects on the fusion assay when added alone at the highest concentration used (100 µg/ml) (data not shown). Serially diluted peptides were preincubated with C-18 (at 20 µg/ml; 1.1 µM) for 1 h at 37 °C. The solutions, containing C-18 mixed with CCR5-derived peptides, were then added to 96-well plates containing PM1 cells for an additional 1 h at 37 °C. 12E1 cells expressing the JR-FL envelope were added to the same wells (at a 1:1 ratio), and syncytia were scored after 3 h. Based on the maximal inhibition seen by C-18 alone, it was possible to calculate the peptide concentrations required for 50% reduction of C-18-mediated fusion inhibition. As can be seen in Fig. 3A, a peptide containing the complete N-terminal sequence (31 amino acids) could reverse the inhibitory activity of C-18 at ID50 of 23.1 µg/ml (85.2 µM). Control peptide derived from the CXCR4 N terminus did not reverse the inhibitory activity of C-18 (Fig. 3E). The CCR5 N-(1–31) was also able to block the binding of 125C-18 or FITC-C-18 to CEM.NKR.CCR5 cells as demonstrated in Fig. 4, left and right panels, respectively.

Because the N-terminal 31 amino acids may acquire secondary structure in solution, and this region of CCR5 was shown to contain multiple distinct binding sites for different agonists, we next tested shorter sequences from the CCR5 N terminus. The last 16 amino acids gave very minimal C-18 blocking (data not shown). On the other hand, a peptide containing only the first 18 amino acids from the N terminus of CCR5 (CCR5 N-(1–18)) was 150-fold more active than the 31-amino acid peptide in reversing the fusion inhibition of C-18 (Fig. 3B). A scrambled peptide, CCR5 N-(1–18)-SCR, had no activity and did not reverse the inhibitory activity of C-18 (Fig. 3F). The observed increased activity of N-(1–18)-peptide compared with N-(1–31)-peptide suggests that the shorter peptide has a better fit for C-18, whereas in the context of the 31-amino acid CCR5 N-terminal peptide, a significant amount of folding may occur in solution resulting in masking or steric hindrance for C-18 binding. The EC-II-derived peptide did not significantly block the fusion inhibitory activity of C-18 (data not shown). To map further the critical amino acids within the CCR5 N-(1–18) required for C-18 activity, we examined the ability of two shorter peptides from this region to compete out C-18 fusion inhibition. Most surprisingly, the first 9 residues of the CCR5 N terminus (CCR5 N-(1–9)) had no competitive activity (not shown), whereas the peptide containing residues 10–18 was very active with ID50 of 0.14 µM, identical to CCR5 N-(1–18) (Fig. 3, B and C).



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  		FIG. 3.
Blocking of C-18 fusion inhibitory activity by preincubation with CCR5-derived soluble peptides. C-18 was used at 20 µg/ml (1.1 µM). C-18 was preincubated with the following peptides: R5 N-(1–31) (panel A); R5 N-(1–18) (panel B); R5 N-(10–18) (panel C); R5 N-(1–18), Tyr-10(S), Tyr-14(S)) (Y10, 14sulf) (panel D); CXCR4 N-(1–38) (panel E); and R5 N-(1–18) scrambled peptide (SCR) (panel F). The % inhibition by C-18 alone ranged between 55 and 75%. Serially diluted peptides were incubated with C-18 in 0.5-ml microcentrifuge tubes for 1 h at 37 °C. The mixtures were added to wells containing PM1 cells for an additional 1 h at 37 °C, after which effector cells expressing JR-FL (or Ba-L) envelope were added (effector/target ratio of 1:1). Syncytia were scored after 3 h. The peptide concentration required to reduce the fusion inhibitory activity of C-18 by 50% was based on at least three separate determinations for each peptide. No fusion inhibition was observed adding the peptides alone under the same conditions (not shown).

 



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  		FIG. 4.
CCR5 N-terminal peptide can block binding of C-18 to CCR5-expressing CEM cells. Left panel, 125I-labeled C-18 at 1 nM concentration was incubated with CCR5+ and CCR5– cells alone or in the presence of a 1:1, 10:1, or 100:1 excess of CCR5 N-(1–31)-peptide (or control peptides, not shown) for 90 min at 4 °C. Cells were then washed, and the bound fraction was measured radioactively. Specific binding was calculated by subtracting the nonspecific background observed with CCR5– cells. Right panel, recombinant FITC-labeled C-18 was incubated with CEM (gray histogram) and CEM.NKR.CCR5 cells (open histogram) for 30 min at 4 °C, either alone or in the presence of CCR5 N-(1–31)-peptide. Cells were then washed, and the bound C-18 was analyzed by flow cytometry. The data shown are representative of two experiments with similar results.

 
It was reported previously that the CCR5 N terminus contains several tyrosine residues that are sulfated, and this post-translational modification is required for interaction with both chemokines and GP120-CD4 complexes (9, 26). Thus, we tested peptide CCR5 N-(1–18) containing two sulfated tyrosines (Tyr-10(S) and Tyr-14(S)). Our data revealed that the sulfated peptide was able to reduce the activity of C-18 with almost identical ID50 values to that of the unsulfated CCR5 N-(1–18)-peptide (Fig. 3D).



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  		FIG. 5.
CCR5 N-(1–18)- and N-(10–18)-peptides block binding of 125I-C-18 to CEM.NKR.CCR5 cells. 125I-Labeled C-18 at 1 nM concentration was incubated with CCR5+ and CCR5– cells alone or in the presence of a 1:1, 10:1, or 100:1 excess CCR5 N-(1–31)-peptide (or control peptides, not shown) for 90 min at 21 °C. Cells were then washed, and the bound fraction was measured radioactively. Specific binding was calculated by subtracting the nonspecific background observed with CCR5– cells. The data shown are representative of two experiments with similar results. SCR, scrambled peptide of CCR5 N-(1–18) (see also Fig. 7).

 



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  		FIG. 6.
C-18 does not inhibit fusion of cells expressing N-terminal truncated ({Delta}2–19) CCR5. HeLa.CD4.CCR5 (JC.6 cells) or HeLa.CD4.CCR5(N{Delta}2–19) (R5d18.2 cells) were treated with C-18 (20 µg/ml) or with mAb 2D7 (20 µg/ml) for 1 h at 37 °C, followed by addition of 12E1 expressing JR-FL envelope. Syncytia were scored after 2 h. Control syncytia values (no inhibitor added) were 984 ± 67 and 940 ± 55 for the two cell lines, respectively. All groups were set in triplicate, and S.D. ranged between 10 and 15% of the means. The experiment represents three experiments with similar results.

 
The N-(1–18)- and N-(10–18)-peptides were also tested for their ability to block the binding of 125I-C-18 to CCR5-expressing CEM cells (Fig. 5). As was demonstrated in the fusion assay, peptides encompassing N-terminal amino acids 1–18 or 10–18 were significantly more effective than the 31-amino acid peptide in competing C-18 binding to CCR5+ CEM cells. Even at 1:1 molar ratios, significant displacement of 125I-C-18 was observed with the shorter N-terminal peptides (Fig. 5). As expected, no inhibition of C-18 binding was seen with a control (1–18, scrambled) peptide (SCR).

C-18 Does Not Block HIV-1 Infection of Cells Expressing N-terminal Truncated CCR5 Co-receptors—To address further the contribution of the N terminus of CCR5 to C-18 binding and fusion inhibitory activity, we tested two recently generated HeLa.CD4 cell derivatives expressing either wild-type CCR5 (JC.6) or a CCR5 with N-terminal truncation of amino acids 2–19 (R5d18.2). In a fusion assay with effector cells expressing the JR-FL envelope, both target cells could fuse well. This assay is very sensitive because the effector cells are infected overnight with vaccinia expressing HIV envelopes, resulting in high envelope-expression levels. It was clear that although mAb 2D7 could block fusion of cells expressing either wild-type or truncated CCR5, C-18 completely lost its inhibitory activity against cells expressing the N-terminal truncated CCR5 molecules (Fig. 6). The same cells were also used in an infection assay by using a variant of JR-CSF that was adapted to grow on R5d18.2 cells. It was reported that this viral variant does interact with the N terminus of CCR5, even though it no longer requires this interaction for fusion and infection (most likely due to increased binding avidity to other CCR5 domains) (40). In our study, the JR-CSF18.2adD2 variant replicated 2-fold better on the JC.6 cells expressing wild-type CCR5 compared with cells expressing truncated CCR5. C-18 reduced infection of JC.6 cells by 75%. In contrast, the same viral variant was resistant to C-18 inhibition when grown on the R5d18.2 cells (Table II). These data further suggested that the first 19 N-terminal amino acid sequence of CCR5 contains crucial residues required for C-18 binding and antiviral activity.


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  		TABLE II
C-18 does not inhibit infection of R5d18.2 HeLa cells expressing CCR5 with truncated N-terminal ({Delta}2-19)

HeLa-CD4.JC.6 and HeLa-CD4R5d18.2 were incubated with C-18 for 2 h at 37 °C. JR-SCF.18.2ad was added to all wells at 50 TCID50 per well (five replicates). Virus/inhibitor was removed after 24 h at 37 °C by extensive washings, and cells were cultured for 10 days. Medium was exchanged daily. p24 values are from day 5 post-infection. The experimental data shown are representative of three experiments performed.

 
Alanine Scanning to Determine Key CCR5 Contact Residues—To map further the amino acids within CCR5 N-(1–18) critical for C-18 activity, we next conducted alanine-scanning mutagenesis, replacing each of the amino acids in the shorter peptide with alanine (Table III). Peptides with alanine replacement of either Tyr-10, Asp-11, or Glu-18 were fully active. On the other hand, alanine substitutions in each of the remaining amino acids (12–17) in this peptide significantly reduced the ability of the peptide to reverse the fusion inhibitory activity of C-18. The largest loss of activity was observed with I12A, Y14A, T16A, and S17A mutant peptides (Table III).


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  		TABLE III
Alanine scanning for mapping of the C-18-binding residues in the CCR5 N-(10-18)-peptide

Serially diluted peptides were incubated with C-18 in 0.5-ml microcentrifuge tubes for 1 h at 37 °C. The mixtures were added to wells containing PM1 cells for an additional 1 h at 37 °C, after which effector cells expressing the JR-FL envelope were added (effector/target ratio of 1:1). Syncytia were scored after 3 h. The peptide concentration required to reduce the fusion inhibitory activity of C-18 by 50% was based on at least three separate determinations for each peptide (see also Fig. 3). In all experiments the control syncytia (in absence of C-18) ranged between 300 and 400 syncytia per well with 10% S.D. The % inhibition by C-18 alone ranged between 65 and 75%. No fusion inhibition was observed on adding the peptides alone under the same conditions (data not shown).

 
These peptides were also tested in a direct binding ELISA.

Wells coated with C-18 were reacted with biotinylated CCR5-(1–18)-peptide or biotinylated scrambled peptide containing the same 18 amino acids. As seen in Fig. 7, using streptavidin-horseradish peroxidase for the detection of bound peptide, it was possible to measure the binding of biotinylated CCR5 N-(1–18)-peptide to C-18 in a dose-dependent manner. No association of the scrambled peptide with C-18 was detected. In the peptide competition ELISA, when the CCR5 N-(10–18)-peptides containing alanine substitutions were added to the C-18-coated plates at 1-, 10-, or 100-fold molar excess, they competed with binding of the biotinylated CCR5 N-(1–18)-peptide in a dose-dependent fashion and in good agreement with the results from the fusion assay, again showing the importance of CCR5 residues 12–17 for C-18 binding (compare Fig. 8 with Table III). The location of the key residues in the N terminus that were shown to be involved in C-18 binding is circled in the CCR5 model in Fig. 9 (according to Siciliano et al. (27)).



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  		FIG. 7.
CCR5 N-(1–18)-peptide binds to C-18. ELISA plates were coated with C-18 and were reacted with serially diluted biotinylated CCR5 N-(1–18)-peptide or with biotinylated scrambled peptide (CCR5 N-(1–18)-SCR) containing the same amino acids, followed by addition of peroxidase-conjugated streptavidin. The reactions were quantified using 2,2'-azinobis-(3-ethylbenzthiazoline sulfonic acid) substrate. Absorbance was measured at 405 nm.

 



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  		FIG. 8.
Competition of C-18 binding to biotinylated CCR5 N-(1–18)-peptide with shorter N-terminal peptides containing single amino acid-alanine substitutions. C-18-coated plates were incubated with synthetic peptides derived from CCR5 N-(1–18), CCR5 N-(1–9), CCR5 N-(10–18), and a series of CCR5 N-(10–18) with alanine substitutions. All peptides were at 10, 1, or 0.1 µM. After 30 min of incubation at room temperature, the biotinylated CCR5 N-(1–18)-peptide was added at 100 nM (competitor/binding peptide ratio of 100:1, 10:1, or 1:1). After 1 h at room temperature, the plates were washed and developed as in Fig. 7.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The protozoan parasite T. gondii cyclophilin produces a protein, cyclophilin-18 (C-18), that can bind to the chemokine receptor CCR5, apparently as a result of a molecular mimicry with the native ligands. In mice, the C-18/CCR5 interaction is involved in the up-regulation of interleukin-12 production, an important mediator of host resistance to this pathogen (15, 28). Although many other pathogens acquired the ability to express chemokine-like proteins or chemokine receptor analogues (29, 30), to our knowledge, only T. gondii expresses a cyclophilin isomer that binds to CCR5 from several diverse species. In the previous study (17), we have identified structural determinants within C-18 required for its antiviral activity. In this counterpart study, we have defined the critical residues within human CCR5 sequence required for binding and HIV inhibitory activity of C-18. Our experiments identified a short sequence in the N terminus of CCR5, amino acids 12–17, as a crucial docking site for C-18. Based on the alignment of CCR5 sequences in mouse, human, and rhesus macaques (Fig. 10), there are five conserved residues between human and mouse CCR5 in this region, including two tyrosines at positions 10 and 14, which help explain C-18 binding to the cell surface CCR5 from various species. The homology with rhesus CCR5 is much higher, with only a single asparagine to aspartic acid change in amino acid 13. This high level of homology in this region would suggest that rhesus CCR5 should support C-18 binding. Experiments are in progress, and preliminary data demonstrate C-18 inhibition of simian immunodeficiency virus envelope-mediated fusion with activated rhesus peripheral blood mononuclear cell consistent with this prediction.

HIV-1 GP120 also evolved to utilize CCR5 as a major co-receptor for viral entry. Extensive work identified a key role for the N terminus and ECL-2 in GP120 binding (reviewed in Ref. 8). More importantly, sulfated synthetic peptides derived from the N terminus of CCR5 were shown to bind to GP120-CD4 complexes in ELISA (31). Furthermore, binding of the natural CCR5 ligands MIP1-{alpha}, MIP1-{beta}, and RANTES (regulated on activation normal T cell expressed and secreted) involves similar but not completely overlapping sites in CCR5. In addition to the N terminus and ECL-2, these {beta}-chemokines were demonstrated to interact with the transmembrane region of CCR5 (25, 32–34). Thus, a common feature of the binding to CCR5 is docking to the less conformationally constrained N terminus, followed by additional interactions with either the EC loops or the transmembrane regions. In our earlier study we demonstrated that MIP1-{beta} and an R5 GP120 (Ba-L) can compete with iodinated C-18 for binding to CEM.NKR.CCR5 cells, suggesting that the binding sites are at least partially overlapping (16).



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  		FIG. 9.
Localization of the main C-18-binding site in the N terminus of CCR5. The thread model of human CCR5 was according to Siciliano et al. (27). The highlighted amino acids are those that differ between human and rhesus CCR5 proteins.

 



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  		FIG. 10.
Alignment of CCR5 sequences from human, rhesus, and mouse.

 
Of interest is the role played by sulfated tyrosines in the N terminus of CCR5 in C-18 binding/activity. It was demonstrated previously that sulfation of tyrosine residues in positions 3, 10, 14, and 15 significantly contributes to the efficiency of GP120 binding and increased viral entry (9, 11, 26). The exact contribution of the sulfate moieties to GP120 binding has not been deciphered, but it was postulated to add a polar, highly charged surface that interacts with positively charged residues in the GP120 V3 or in the co-receptor-binding {beta}-sheet. In our study, CCR5 N-(1–18)-peptides containing either sulfated (Tyr-10(S) and Tyr-14(S)) or unsulfated tyrosines were equally potent in binding to C-18 in solution and blocking its subsequent ability to inhibit fusion. In this regard, it is not yet clear what role is played by positively charged C-18 amino acids in CCR5 binding.

Several safety concerns need to be considered for therapeutic agents targeting self-receptors. Unregulated signaling, induction of proinflammatory cytokines, and receptor down-regulation and/or interference with key biological functions must be addressed. In the case of C-18, we established previously that it does not induce down-regulation of surface CCR5 (16). In our more recent study of the structural determinants of the anti-HIV activity of C-18, it was also found that several mutations designed to destroy completely the enzymatic activity of C-18 did not reduce its HIV-1 blocking activity. More importantly, the same mutations did abolish the ability of C-18 to induce interleukin-12 from mouse dendritic cells (17). Thus, these mutants are likely to have a higher safety profile for future human trials. These and other C-18 derivatives are currently being tested in vitro for their anti-HIV and anti-simian immunodeficiency virus activities.

Several studies demonstrated that early depletion of CD4+ CCR5+ T cells in gut-associated tissues occurs early and at all stages of HIV-1 infection, even in the face of highly active antiretroviral therapies. It was suggested that antiviral therapies may be augmented by new interventions aimed at blocking chemokine receptor-mediated fusion targeting the mucosal immune system where the vast majority of CD4+CCR5+ T cells reside. Further function/structure studies on the interaction of C-18 with CCR5 could be applied toward the synthesis of either more potent C-18 derivative or a C-18 mimic (peptides or small molecules) that will be of potential use as a microbicidal agent, either alone or in combination with other CCR5- and HIV-blocking compounds (35–39).


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Intramural AIDS Targeted Antiviral Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} Supported by the Network of Inflammation Research funded by the Swedish Foundation Strategic Research. Back

§ To whom correspondence should be addressed. Tel.: 301-827-0784; Fax: 301-496-1810; E-mail: goldingh{at}cber.FDA.gov.

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; HIV, human immunodeficiency virus; ECL-2, second extracellular loop; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; WT, wild type; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium. Back


   ACKNOWLEDGMENTS
 
We are grateful to Keith Peden, Carol Weiss, and Paolo Lusso for their helpful suggestions during the preparation of this manuscript. We thank Jody Manischewitz for technical support.



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