Hiv-1 exploits ccr5 conformational heterogeneity to escape inhibition by chemokines

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Philippe COLINM=§, Yann BENUREAUM§, Isabelle STAROPOLIM, Yongjin WANGM, Nuria Gonzalez, Jose ALCAMI, Oliver HARTLEY, Anne BRELOTM, Fernando ARENZANA-SEISDEDOSM, and Bernard LAGANEM

Author Affiliations:

M INSERM U819, Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France.

= Univ. Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Rue du Docteur Roux, 75015 Paris, France.

Instituto de Salud Carlos III, 28220-Majadahonda, Madrid, Spain.

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland.
Corresponding Author: Bernard Lagane, Institut Pasteur, 28 rue du docteur Roux, 75724, Paris cedex 15, France. Tel.:+33145688945. Fax:+33145688941.

§ PC and YB contributed equally to this work.


CCR5 is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4+ T-lymphocytes. Chemokines exert anti-HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G-proteins (NFG-proteins). While native CCR5 chemokines bind with subnanomolar affinity to NFG-protein-coupled CCR5, gp120/HIV-1 does not discriminate between NFG-protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G-protein independent, suggesting that ‘low-chemokine affinity’ NFG-protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC50 values for chemokine-mediated endocytosis reflecting their low-affinity constant value for NFG-protein-uncoupled CCR5. Abolishing CCR5 interaction with NFG-proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed new light into the inhibitory mechanisms of anti-HIV-1 chemokine analogs and provide insights for the development of new anti-HIV molecules.


CCR5 is the principal coreceptor for entry of human immunodeficiency virus type-1 (HIV-1), used together with CD4 to enter and infect target cells (1), and a receptor for agonist (CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES) and antagonist/weak partial agonist (CCL7/MCP-3) chemokines (2, 3). The native agonist chemokine ligands of CCR5 induce conformational changes in the receptor that promote activation of pertussis toxin (PTX)-sensitive, heterotrimeric  G-proteins (Gi/o-type G-proteins) by catalyzing an exchange of GTP for GDP on the G subunit. The GTP-bound G subunit and the G dimer then trigger intracellular signaling pathways involved in chemotaxis and activation of leukocytes (4).

Native CCR5 chemokines inhibit infection of R5-tropic HIV-1 in vitro. This occurs via two mechanisms: sterically preventing the viral envelope gp120 from binding to the coreceptor and reducing cell surface coreceptor levels by inducing receptor downregulation (5-7). They are secreted by a number of cell types and in particular immune cells including R5 HIV-1 target cells (6, 8, 9). The potential role of native CCR5 chemokines in blocking HIV-1 transmission and progression has been extensively studied (9-12), but their efficacy as protective factors remains a matter of debate (13, 14). A major paradox relates to the observation that native CCR5 chemokines show lower antiviral potencies than would be expected based on their CCR5 binding affinity constants (15-18), which are in the subnanomolar range (2, 19, 20), much lower than the corresponding value for the HIV-1 envelope glycoprotein gp120, which is approximately 10 nM (19, 21).

A number of CCR5 chemokine analogs with improved antiviral potency have been identified, including N-terminally modified RANTES analogs with agonist (AOP-, PSC- or 6P4-RANTES) or antagonist features (5P12- or 2P3-RANTES), which represent promising molecules as topical microbicides (18, 22). While the enhanced potency of agonist analogs can be explained in terms of their increased capacity to induce CCR5 downregulation (23), the inhibitory mechanism of antagonist analogs, which neither activate G protein signaling nor induce receptor downregulation, is more elusive. It was speculated that it might involve increased steric blockade of CCR5, but competition binding assays using labeled CCL4 as a tracer did not show any significant increase in CCR5 binding affinity (22).

In this study, we present evidence that conformationally different CCR5 subpopulations with distinct chemokine binding capacities are present at the surface of HIV-1 target cells. In particular, a fraction of receptors shows strikingly low binding affinity for native CCR5 chemokines, providing an explanation for why native CCR5 chemokines have unexpectedly low anti-HIV-1 potencies. Our results also shed further light on the inhibitory mechanism of chemokine analogs, showing that they overcome the challenge of the chemokine low-affinity CCR5 population through (i) more efficient receptor downregulation and/or (ii) increased binding affinity for gp120-binding receptors. Overall, these findings explain how R5 HIV-1 could escape from inhibition by native CCR5 chemokines in the course of infection and provide clues for the development of new chemokine analogs as HIV inhibitors.

Distinct CCR5 populations are differentially utilized by HIV-1 gp120 and chemokines. Chemokines and the CD4-bound form of HIV-1 envelope glycoprotein gp120 competitively bind to overlapping regions of CCR5 (24). Thus, to investigate whether distinct CCR5 populations interact differently with chemokines and gp120, we first tested unlabeled native chemokines (CCL-3, -4, -5 or -7) or chemokine analogs (AOP-, PSC-, 2P3-, 5P12-, or 6P4-RANTES) for their ability to inhibit binding of either 125I-labeled CCL3 (125I-CCL3) or the 35S-labeled gp120 from the HIV-1 primary strain Bx08 (35S-gp120Bx08) on membranes from CCR5-expressing HEK 293T cells (HEK-R5 cells) (Fig. 1, Table 1). Using the data obtained, we calculated the affinity constant values (Ki) of the competing ligands for receptors using the Cheng and Prusoff equation (see SI materials and methods).

Except for CCL7, displacement of 125I-CCL3 binding revealed high affinities of competitors for CCR5, with Ki values in the nM range or lower (Table 1). The Ki values obtained for the native CCR5 agonists CCL-3, -4 and -5 are comparable to the KD values determined for these ligands in saturation binding assays (Table 1), consistent with binding of these chemokines to a similar class of high-affinity receptors in both competition and saturation assays. In contrast, CCL-3, -4 and -5, as well as the chemokine analogs AOP- and PSC-RANTES, only partly displaced 35S-gp120Bx08 binding when used at a 100 nM concentration (Fig. 1B), suggesting that they have a lower affinity for 35S-gp120Bx08-binding receptors as compared to 125I-CCL3-binding receptors. In contrast, the observation that 35S-gp120Bx08 binds marginally to CCR5 in the presence of 100 nM 2P3-, 5P12- or 6P4-RANTES (Fig. 1B) suggests that these chemokine analogs preserve high affinity interactions with 35S-gp120Bx08-binding CCR5.

In dose-response experiments, 2P3-, 5P12- or 6P4-RANTES and 35S-gp120Bx08 competed for binding to an apparent single class of receptors, as is revealed by monophasic competitive binding curves (Fig. 1C-D). The Ki values in the nM range calculated for these chemokine analogs confirm high-affinity interactions with the 35S-gp120Bx08-binding receptors (Table 1). CCL7 similarly bound to a single class of 35S-gp120Bx08-binding CCR5 for which the affinity of the chemokine was higher than that for 125I-CCL3-binding CCR5 (Ki = 34 vs 119 nM). In contrast, displacements of 35S-gp120Bx08 binding by the agonists CCL3 (Fig. 1C), CCL4 and PSC-RANTES (Fig. 1D) gave biphasic curves, consistent with the presence of two distinct 35S-gp120Bx08 receptor populations, one with high affinity for these chemokines, the other with significantly lower affinity. CCL-3, -4 and PSC-RANTES had Ki values for interaction with the ‘high-chemokine affinity’ receptor population similar to those determined in 125I-CCL3 displacement assays (Table 1), suggesting that the ‘high-chemokine affinity’ population of 35S-gp120Bx08-binding receptors and 125I-CCL3-binding receptors represent the same receptors. Interestingly, the Ki values obtained for the ‘low-chemokine affinity’ CCR5 population range from a few tens of nM up to more than 10-6 M (Table 1), thus exceeding the KD value for the interaction of 35S-gp120Bx08 with CCR5 determined in saturation assays (≈ 10 nM (19)). Hence, this fraction of CCR5 has a higher affinity for gp120 than for native CCR5 chemokines.

Poor ability of native CCR5 chemokines to displace gp120 binding to CCR5 correlates with low antiviral activity. We next investigated whether low anti-HIV potency of native chemokines is related to low-affinity interactions with gp120-binding receptors. For that purpose, we infected activated CD4+ T-lymphocytes, which represent the major target cells for R5 HIV-1, and HeLa P4C5 cells with infectious Bx08Ren viruses expressing gp120 from the Bx08 strain in the presence of native CCR5 chemokines or chemokine analogs (Table 1, Fig. S1A and S1B). Except for the chemokine analog PSC-RANTES, the antiviral potencies of all other chemokines correlated better with their ability to displace the binding of 35S-gp120Bx08 than that of 125I-CCL3. In particular, the native CCR5 agonists (CCL-3, -4 and -5) that show low-affinity interactions with a proportion of gp120-binding receptors had much more weaker antiviral activities than chemokine analogs, even though both groups of molecules have comparable affinities for 125I-CCL3-binding CCR5 (Table 1). Similar results were obtained using five other NL4-3Ren-derived viruses expressing R5 gp120 sequences from laboratory-adapted (JRRen) as well as primary (25Ren, 34Ren, 50Ren or 58Ren viruses) viruses (Fig. S2).

The potent antiviral activities of the antagonist RANTES analogs 5P12 and 2P3, which do not downregulate CCR5 (Fig. 5B and ref. (22)), reflect their increased binding affinities detected using 35S-gp120Bx08 as a tracer (Fig. 1). The enhanced potency of PSC-RANTES, which occurs despite its relatively low capacity to compete with 35S-gp120Bx08 for binding to CCR5, is likely to be due to its enhanced capacity to induce CCR5 downregulation (23). To test this hypothesis, we performed infection inhibition experiments under conditions where receptor downregulation is suppressed. HeLa P4C5 cells were pre-incubated for 2 h with PSC-RANTES (40 nM), 5P12-RANTES (40 nM) or the CCR5 inverse agonist maraviroc (MVC, 20 M) at either 37°C or at 4°C, a temperature at which receptor endocytosis does not occur. Bx08Ren virus was then added to the cells, which were incubated for a further 2 h at 4°C, then washed in cold PBS, warmed to 37 °C for 15 min to allow entry of attached viruses, trypsin-treated to remove residual viruses and incubated for 48 h at 37 °C (Fig. 2). Under conditions where CCR5 downregulation is suppressed, the antiviral activity of PSC-RANTES was almost completely abrogated, but the inhibitory potency of 5P12-RANTES or MVC was unaffected, in accordance with our results in Fig. 1 showing that PSC- and 5P12-RANTES are weak and potent inhibitors of gp120 binding to CCR5. Hence unlike 5P12-RANTES and maraviroc, PSC-RANTES owes a large part of its inhibitory activity to its capacity to induce CCR5 downregulation. Importantly, these results also validate the notion that the receptors interacting with monomeric gp120/soluble CD4 complexes in the binding assays presented here (Fig. 1B-D) and those that are used by infectious virus particles at the surface of intact cells in infection assays extensively overlap and would represent similar receptor populations.

CCR5 coupling to nucleotide-free G-proteins differentially regulates native agonist chemokine and gp120 binding. It has been established that conformations of GPCRs with high-affinity for agonists are stabilized by coupling to guanine nucleotide-free G-proteins (NFG-proteins), and that the receptors are induced to shift towards low-affinity conformations as soon as G-proteins are occupied by nucleotides (25). Similarly to what we and others showed in mammalian cell lines (19, 26), we observed that CCR5 coupling to NFG-proteins also stabilizes the receptor in a high affinity conformation for agonists in HIV-1 target cells. Indeed, the non hydrolysable GTP analogs GTPS and Gpp(NH)p or PTX, which inactivates Gi/o-proteins, decreased 125I-CCL3 binding to CCR5 expressed in human lymphoblastoid CD4+ T-cell lines (A3.01-R5 cells) or primary T-lymphocytes to levels approaching that of non specific binding (Fig. 3A). Saturation binding of 125I-CCL3 to membranes from HEK-R5 cells further revealed that Gpp(NH)p decreases the maximum number of binding sites for the chemokine (Bmax) from 10.71.3 to 2.90.7 pmole/mg of protein (Fig. 3B) while only slightly affecting the KD value from 0.250.05 to 0.460.08 nM, indicating that Gpp(NH)p reduces the amount of receptors that are of high affinity for 125I-CCL3. In line with this, 0.1 nM 125I-CCL3 showed only background levels of binding to membranes from HEK cells expressing the R126N-CCR5 mutant, which does not activate G-proteins (27) (Fig. 3D). Similarly to CCL3 and CCL4 (26), high-affinity binding of CCL5 also required CCR5 coupling to NFG-proteins (Fig. 3C).

In contrast to native CCR5 agonist chemokines, R5 HIV-1 gp120 acts as an antagonist/weak partial agonist for CCR5 as it does not discriminate between NFG-protein-coupled or uncoupled CCR5 and binds equally well to R126N-CCR5 and wild-type CCR5, both in the presence and absence of Gpp(NH)p (Fig. 3E). This led us to hypothesize that the biphasic competitive binding curves obtained with native CCR5 agonist chemokines using 35S-gp120Bx08 as a tracer is a reflection of (i) the existence of populations of both NFG-protein-coupled and NFG-protein-uncoupled receptors with respectively high and low affinity for these chemokines, and (ii) the capacity of gp120 to bind indiscriminately to either population. We tested this hypothesis by repeating the competition experiments of 35S-gp120Bx08 binding by CCL3, in the presence and absence of Gpp(NH)p (Fig. 3F). Treatment with Gpp(NH)p decreased the proportion of ‘high-chemokine affinity’ receptors versus ‘low-chemokine affinity’ receptors from 43% to 16% (p = 0.016 in unpaired, two-tailed student’s t test), without affecting the Ki value of the ‘low-chemokine affinity’ receptor population (Table 1, Fig. 3F). This result is consistent with our previous observation that Gpp(NH)p eliminates the fraction of 35S-gp120Bx08-binding CCR5 that binds CCL4 with high affinity (19).

Chemokine-mediated inhibition of HIV infection and CCR5 endocytosis are G-protein independent processes. Based on the observation that HIV envelope binds indiscriminately to ‘high-chemokine affinity’ NFG-protein-coupled CCR5 and ‘low-chemokine affinity’ NFG-protein-uncoupled receptors, we hypothesized that infection in the presence of chemokine ligands would be more likely to occur via the low-chemokine affinity NFG-protein-uncoupled receptors, and that the chemokine ligands would be required to engage this population of receptors in order to achieve inhibition of infection. This would explain why native CCR5 agonist chemokines have low potency as HIV inhibitors.

To test this hypothesis, we treated activated CD4+ T-cells, A3.01-R5 or HeLa P4C5 cells with PTX and then infected them with R5 HIV-1 in the presence of native CCR5 chemokines or chemokine analogs (Fig. 4, S2 and S3). PTX attenuated 125I-CCL3 binding to target cells (Fig. S3A and 3A) and abrogated chemokine-induced chemotaxis (Fig. 4A), indicating that CCR5 coupling with Gi/o-proteins is required for both high-affinity binding of the ligands and signal transduction. In contrast, PTX changed neither viral infectivity (Fig. S2) nor the potency of chemokines to block infection (Fig. S2, S3B, 4B and Table 1). This suggests that CCR5 engagement by HIV-1 is independent of G-proteins and that high-affinity binding of ligands to NFG-protein-coupled CCR5 does not make a significant contribution to their capacity to inhibit infection.

These results also imply that CCR5 downregulation, which contributes to the anti-HIV activity of agonist chemokines, is not dependent on engagement of high-chemokine affinity NFG-protein-coupled CCR5. To address this possibility, we tested the ability of CCL-3, -4, PSC-, 6P4- and 5P12-RANTES to downregulate FLAG-tagged CCR5 in HEK 293 cells (28) with and without PTX, which decreases high affinity binding of 125I-CCL3 by 87.4 ± 11.8 % (Fig. 5A-C, Table S1). PTX influenced neither the efficacy nor the potency (EC50) of chemokines to downregulate CCR5 (Fig. 5A-B, Table S1). Moreover, the kinetic rates of CCR5 downregulation induced by PSC-RANTES and CCL4 were unchanged by PTX treatment (t1/2 (min) = 3.3 ± 0.6 vs 3.2 ± 0.9 and 8.9 ± 0.8 vs 8.7 ± 1.7 for PSC and CCL4 in the absence or in the presence of PTX, respectively) (Fig. 5C). Hence interaction with high-chemokine affinity NFG-protein-coupled CCR5 is not a requirement for the induction of CCR5 downregulation by its ligands.
CCR5 downregulation involves low-affinity interactions of native CCR5 agonists with internalizing receptors. PSC- and 6P4-RANTES had nanomolar EC50 values for CCR5 downregulation (Table S1), while the EC50 values for CCL-3 and -4 exceeded by more than 2 or 3 orders of magnitude their Ki value for NFG-protein-coupled CCR5 (Tables 1 and S1). The differential abilities of chemokines to downregulate CCR5 could be due to internalization-competent CCR5 that might represent a receptor subpopulation to which CCL-3 and -4, but not PSC- and 6P4-RANTES, bind with a low affinity. Alternatively, but not exclusively, RANTES analog-induced inhibition of receptor recycling could also contribute to their potent ability to downregulate CCR5, as previously suggested (23).

To assess these hypotheses, we compared the abilities of PSC-RANTES and CCL4 to downregulate WT-CCR5 or the 349-CCR5 mutant, which does not recycle back to the cell surface (28) (Fig. 5D). As compared to WT-CCR5, CCL4 downregulated 349-CCR5 with higher potency (6-fold, Table S1) and efficacy, confirming that receptor recycling interferes to some extent with the ability of CCL4 to downregulate CCR5. PSC-RANTES also downregulated 349-CCR5 more efficiently than WT-CCR5 (93.9 ± 1.6 % vs 72.6 ± 3.3 %, respectively), albeit with comparable potencies (Table S1), suggesting that PSC-RANTES slows down CCR5 recycling but does not prevent it. However, CCL4 induced endocytosis of 349-CCR5 with a 18-fold higher EC50 value as compared to PSC-RANTES, indicating that low potency of CCL4 in downregulating CCR5 is modestly due to its inability to prevent receptor recycling. Rather, this EC50 value for endocytosis of 349-CCR5 by CCL4 (47 nM) is similar to its Ki value for interaction with the ‘low-chemokine affinity’ NFG-protein-uncoupled population of CCR5 (44.7 nM). This suggests that native CCR5 agonist chemokines have a low potency to downregulate CCR5 owing to their inability to prevent CCR5 recycling and, above all, to their low affinity for NFG-protein uncoupled CCR5 undergoing endocytosis.


Our findings indicate that inhibition by native CCR5 chemokines of HIV-1 infection is hindered by a proportion of receptors that exists in a low-chemokine affinity conformation at the target cell surface. This likely explains the discrepancy between the apparently high CCR5 affinities measured previously for native chemokine ligands (2, 20) and their relatively modest potency as entry inhibitors (15-18). Different CCR5 conformations with distinct pharmacological and antigenic properties have been described (27, 29). Here, we found that the apparent affinity of native chemokines and RANTES analogs for CCR5 varies depending on whether 125I-CCL3 or 35S-gp120 is used as a tracer in competition experiments (Fig. 1), identifying that distinct receptor populations interact with 125I-CCL3- and 35S-gp120-binding receptors. Indeed, we further showed that while high-affinity binding of 125I-CCL3 requires CCR5 to be coupled to NFG-proteins, 35S-gp120 binds with the same affinity to both high-chemokine affinity NFG-protein-coupled CCR5 and low-chemokine affinity NFG-protein-uncoupled CCR5. Although native CCR5 agonist chemokines interact with subnanomolar affinities with 125I-CCL3-binding receptors, they bind to the low-chemokine affinity population of 35S-gp120-binding receptors with affinities lower than those of primary gp120 (legend of Fig. 3E), thereby contributing to limiting their antiviral potency.

PTX treatments of HIV-1 target cells had no effect on virus entry and replication (Fig. S2), suggesting that similarly to gp120, HIV-1 attachment to CCR5 is independent of G-proteins, in agreement with our previous data showing that the non-G-protein coupling mutant receptor R126N-CCR5 supports HIV entry (30). The observations that CCR5 is constitutively active (19, 27) and that preformed receptor/G-protein complexes exist in living cells (31) suggest that an equilibrium may exist between NFG-protein coupled and uncoupled CCR5 in HIV target cells. On the other hand, NFG-proteins that stabilize high-agonist affinity conformations of CCR5 likely represent a minor fraction of total G-proteins in intact cells (25). In line with this, our observations that PTX does not change the anti-HIV potency of chemokines (Table 1) suggest that high-chemokine affinity NFG-protein-coupled receptors play a minor role in the antiviral activity of chemokines and that low-chemokine affinity NFG-protein-uncoupled CCR5 represent a portal for HIV entry into target cells. Interaction with NFG-protein-uncoupled CCR5 could allow HIV to evade inhibition by the chemokines secreted in the surrounding environment. At the same time, through high affinity interactions with receptors coupled to NFG-proteins, these chemokines would still be capable of activating target cells, facilitating viral replication (32), and recruiting target cells into sites of HIV replication.

While it is commonly accepted that coreceptor downregulation contributes to chemokine inhibition of HIV-1 infection (5-7), we showed that native CCR5 agonist chemokines exhibit a weak ability to downregulate CCR5, as is indicated by EC50 values for CCR5 downregulation by the chemokines that are close to their Ki values for interaction with the ‘low-chemokine affinity’ population of CCR5. Preventing CCR5 recycling only modestly increases the ability of CCL4 to downregulate CCR5, but several observations suggest that CCR5 downregulation involves low-affinity interactions of native chemokines with NFG-protein uncoupled CCR5. Indeed, we previously demonstrated that R126N-CCR5 does not trigger G-protein signaling but retains -arrestin-dependent endocytosis, indicating that both processes are independent functions of CCR5 mediated by different receptor conformations (27). R126N-CCR5 is also altered in its ability to bind 125I-CCL3 (Fig. 3D) and 125I-CCL4 (26), indicating that the NFG-protein-coupled conformation of CCR5 required for high affinity binding of agonist chemokines is distinct from the CCR5 conformation undergoing endocytosis. This conclusion agrees with our present results that PTX that inhibits CCR5/G-protein coupling and high affinity binding of native agonist chemokines preserves CCR5 endocytosis (Fig. 5). Overall, these data support the view that natural agonist chemokines engage low-affinity interactions with internalizing CCR5, hence explaining why they are weak inducers of CCR5 endocytosis and inhibitors of HIV infection.

Structurally different agonists can stabilize distinct receptor conformations with distinct signaling outcomes (33). In particular, ligands referred to as biased ligands differentially stimulate G-protein- and -arrestin-dependent signaling pathways (34). Similarly, PSC-RANTES and CCL4 have comparable binding affinities for CCR5 (Fig. 1) and potencies for activating G-proteins in a 35S-GTPS binding assay (EC50 = 4.1±0.9 and 6.3±0.4 nM for CCL4 and PSC, respectively, n = 2), while PSC-RANTES is substantially more potent in internalizing CCR5 (Fig. 5), suggesting that the two ligands stabilize distinct CCR5 conformations. In fact, the EC50 value for PSC-RANTES to downregulate CCR5 is roughly equal to its Ki value for interaction with NFG-protein-coupled, 125I-CCL3-binding CCR5, suggesting that PSC-RANTES preserves high-affinity interactions with internalizing CCR5, despite the fact that these receptors are not coupled to NFG-proteins. It could be that PSC-RANTES stabilizes a -arrestin-coupled conformation of CCR5 for which it maintains a high affinity, similarly to other receptors, which are in a high affinity state for agonists when complexed with arrestins (35). Finally, the robust CCR5 downregulation induced by PSC-RANTES explains why the molecule preserves a strong antiviral activity in spite of having a low affinity for gp120-binding receptors. Indeed, preventing CCR5 endocytosis virtually abrogates PSC-RANTES-mediated inhibition of HIV infection (Fig. 2), indicating that steric inhibition of gp120 binding to CCR5 plays a marginal role in the antiviral activity of PSC-RANTES.

The antagonists 5P12- and 2P3-RANTES appeared instead to act solely by potently blocking the interaction between gp120 and CCR5 (Fig. 1). Using these antagonists together with CCR5-internalizing molecules such as PSC-RANTES could in principle represent an interesting therapeutic perspective, albeit no studies have shown yet whether these different analogs have additive inhibitory effects in HIV infection. Interestingly however, we showed here that 6P4-RANTES resembles both 5P12- and PSC-RANTES in that it preserves high affinity for gp120 binding receptors (Fig. 1C) and downregulates CCR5 at nanomolar concentrations. Considered altogether, these results are consistent with 5P12-, 6P4- and PSC-RANTES stabilizing different CCR5 conformations. In line with this, mutations in the transmembrane domains of CCR5 were found to modulate in different ways their ability to inhibit HIV infection, indicating that they have different structural constraints for HIV-1 inhibition (36). Notably, these mutations did not change the ability of the RANTES analogs to inhibit 125I-CCL3 binding to CCR5 (36), again strengthening the notion that chemokines have different structural requirements for interacting with NFG-protein coupled, CCL3-binding receptors and inhibiting gp120 binding and HIV infection.

Overall, our findings document that both mechanisms whereby native CCR5 chemokines exert their anti-HIV activity, inhibition of gp120/CCR5 interactions and CCR5 downregulation, are strongly limited by virtue of their low-affinity interactions with a proportion of CCR5 conformations. Overcoming these limitations explains why RANTES analogs show improved antiviral potencies as compared to their natural counterparts and should help guide the development of new anti-HIV agents. Finally, these limitations could make it difficult to accomplish the blockade of R5 HIV-1 isolates by chemokines in vivo and contribute to their preferential transmission and propagation in the early stages of infection.
Materials and Methods

Information regarding materials (chemokines, HIV-1 glycoproteins, viruses and cells) and experimental procedures (radioligand binding, chemotaxis, receptor downregulation and infection inhibition assays) is provided in SI materials and Methods.


This work was supported by ANRS, SIDACTION, INSERM, Institut Pasteur, the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence “Integrative Biology of Emerging Infectious diseases” (Grant n°ANR-10-LABX-62-IBEID) and the Spanish Ministry of Economy and Competitiveness (FIS PI 080752). OH acknowledges support from the Swiss National Science Foundation. YB, YW and NG were supported by fellowships from SIDACTION, ANRS and the Spanish AIDS Research Network (ISCIII-RETIC RD06/0006), respectively.

Author contributions

P.C., Y.B., I.S., Y.W. and N.G. performed experiments and analyzed data. O.H., J.A. and A.B. contributed materials and technical support. F.A.S. and B.L. designed research and analyzed data and B.L. wrote the manuscript. All authors reviewed and edited the manuscript.


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Table 1. CCR5 binding affinity constants of native chemokines and RANTES analogs and their half-maximal inhibitory concentrations (IC50) of HIV-1 infection in CD4+ T-cells and HeLa P4C5 cells






- Gpp(NH)p

+ Gpp(NH)p













Hi- 0.6±0.1 (43.6±9.7%) Lo- > 1000

Hi - 0.58±0.4 (16.4±6.6 %)

Lo - 597±185.4




> 1000

> 1000


0.37 (a)


± 0.1

Hi- 0.43±0.1 (25±3 %)

Lo- 44.7±6.5

und (b)

Lo - 86±11 (b)













± 1.1

> 1000




> 1000

> 1000




± 31.7



> 1000


> 1000

> 1000




± 0.18










± 0.95

Hi- 1.47±0.03 (31.7±0.5 %)

Lo - 215±35.7













± 0.11











± 0.13














± 0.02










KiCCL3 and Kigp120 represent the equilibrium dissociation constants for interaction of chemokines with CCR5 determined in competition assays using either 125I-CCL3 or 35S-gp120 as a tracer, respectively. KD values are the equilibrium dissociation constants of radiolabeled chemokine-CCR5 complexes deduced from saturation binding experiments. (a) The reported KD value is from ref. (20). (b) The reported Ki values for interaction of CCL4 with 35S-gp120-binding CCR5 in the presence of Gpp(NH)p is from ref. (19). Except for inhibition of infection of T-cells by CCL3 or 6P4 in the presence of PTX, which was performed once, values represent means ± SD of at least three independent determinations. The independent experiments in CD4+ T-cells represent experiments run in cells obtained from different donors. Und, undetectable.


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