LD78beta , A Non-allelic Variant of Human MIP-1alpha (LD78alpha ), Has Enhanced Receptor Interactions and Potent HIV Suppressive Activity*

Robert J. B. NibbsDagger , Jinying Yang§, Nathaniel R. Landau§, Jian-Hua Maoparallel , and Gerard J. GrahamDagger **

From the Dagger  Beatson Institute for Cancer Research, parallel  Department of Medical Oncology, CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD Scotland, United Kingdom and the § Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York 10016

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha ), MIP-1beta , RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha ), termed LD78beta or MIP-1alpha P, has enhanced receptor binding affinities to CCR5 (~6-fold) and the promiscuous beta -chemokine receptor, D6 (~15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alpha P is responsible for this enhanced activity. Moreover, MIP-1alpha P is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alpha P was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alpha P may be of importance in controlling viral spread in HIV-infected individuals.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines are a family of structurally and functionally related proteins that play a central role in the regulation of hemopoietic cell migration during development, immune surveillance, and the establishment of inflammatory and immune responses (1, 2). These biological effects are mediated through a family of cell surface G-protein-coupled heptahelical receptors (3). Recently, these receptors have been implicated in HIV1 pathogenesis with the discovery that a large number of the members of this family are able to act as cofactors for the entry of HIV into cells in vitro (for reviews, see Refs. 4 and 5). The ligands for these receptors are able to compete for binding with the HIV envelope protein to abrogate viral entry (6-8). While an in vivo role for many of these receptors has yet to be demonstrated in the context of HIV infection, CCR5, a receptor for RANTES (regulated on activation, normal T cell expressed and secreted), MCP2 (monocyte chemotactic protein-2), and MIP-1alpha and -beta (macrophage inflammatory protein-1alpha and -1beta ) appears to be critical in HIV pathogenesis as individuals who are homozygous or heterozygous for a CCR5 null mutation are relatively resistant to HIV infection and progression to AIDS, respectively (9-11). Moreover, the CCR5 ligands are produced by activated HIV-specific cytotoxic and helper T cells, whereby they are likely to play an important role in controlling viral spread (12-16). Current data show RANTES to be the most effective antagonist of CCR5-mediated HIV entry, with MCP2 and MIP-1alpha and -1beta being less effective (17-19).

We have been interested in characterizing receptors mediating the biological effects of MIP-1alpha , a member of the beta  subfamily of chemokines able to induce chemotaxis of many mature leukocyte types (1, 2) and a potent inhibitor of hemopoietic stem cell proliferation (20, 21). Mice lacking this gene display dramatic alterations in responses to several infectious agents (22) likely due to leukocyte chemotaxis abrogation. We have recently cloned several receptors for this beta  chemokine from mouse and human sources (23, 24) and found a number of discrepancies with respect to their interaction with murine MIP-1alpha and its presumed human homologue, LD78alpha . Thus, while the murine and human forms of the promiscuous beta -chemokine receptor D6 (23, 24) bind murine MIP-1alpha with high affinity, LD78alpha interacts poorly with these receptors. Similarly, murine CCR5 binds murine MIP-1alpha with high affinity but does not recognize the putative human homologue.

The basis for this marked selectivity has not previously been addressed, however, it is notable that, while the murine genome contains only one copy of MIP-1alpha and -beta , in humans these two genes have been duplicated and mutated to produce two different non-allelic isoforms which still retain >90% homology (25, 26).2 For MIP-1alpha , these have been called LD78alpha and -beta , with LD78alpha being the predominant experimentally used isoform and the one shown to interact poorly with the cloned chemokine receptors. Both forms are transcribed (26) and can be secreted from mammalian cells (6, 21, 27, 28). Interestingly, consideration of differences in the putative signal sequences of the two LD78 isoforms by predictive algorithms,2 suggests that there is variation in the site of signal peptidase cleavage between the two isoforms during secretion, and that LD78alpha is in fact produced without the four anticipated amino-terminal amino acids, ASLA (Fig. 1). Indeed, human MIP-1alpha has been shown to be produced naturally by CD8+ T cells as a -4 variant with the amino-terminal sequence ADTPT (6, 27). We have worked extensively with the LD78beta isoform (21, 28), and numerous amino-terminal sequencing exercises have consistently revealed a "full-length" amino terminus APLAADTPT.

Using full-length and truncated variants of LD78alpha and -beta , we show here that the murine/human MIP-1alpha binding discrepancies are resolved by studying the properties of LD78beta which consistently behaves more like murine MIP-1alpha than does LD78alpha . It appears therefore that LD78beta more accurately represents the functional human homologue of murine MIP-1alpha and that LD78alpha should be considered to be a related but functional distinct chemokine. Importantly, we also show that LD78beta is the most potent natural CCR5 agonist described to date. Furthermore, LD78beta exhibits a much greater ability to antagonize HIV entry through CCR5 than other forms of human MIP-1alpha . In fact, LD78beta is consistently better at HIV1 antagonism than RANTES, previously described as the most potent CCR5-dependant HIV1 entry inhibitor. We demonstrate that the enhanced activity of LD78beta is solely due to the presence of a proline residue at position 2 of the mature protein. We propose renaming LD78alpha and -beta to MIP-1alpha S and MIP-1alpha P, respectively, to reflect the importance of this residue in the functional differences between these two proteins.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Chemokines-- MIP-1alpha S is "human MIP-1alpha " purchased from Peprotech, London, United Kingdom. It is derived from the LD78alpha cDNA and has the amino-terminal sequence of ASLAADTPT. MIP-1alpha S-4 is human MIP-1alpha purchased from R&D Systems, Abingdon, UK. It is derived from the LD78alpha cDNA and starts as ADTPT. MIP-1alpha P is derived from the LD78beta cDNA and was prepared as described previously (28). Sequencing revealed APLAADTPT at the amino-terminal. MIP-1alpha P-4 was produced from a modified LD78beta cDNA in bacteria3 with the amino terminus of ADTPT.

Receptor Binding Studies-- CHO cells expressing chemokine receptors were prepared, and binding assays performed, as outlined previously (23). In short, binding assays were performed using 125I-labeled murine MIP-1alpha at a constant concentration of 600 pM (for D6), 3 nM (for CCR1), or 9 nM (for CCR5), while varying the concentration of unlabeled human MIP-1alpha competitor protein. Remaining radioactivity bound after 90 min, and three ice-cold phosphate-buffered saline washes, was determined. Each point was done in triplicate, the average taken, and converted into a percentage of radioactivity bound in the absence of any unlabeled competitor chemokine. Data were analyzed using the LIGAND software (29).

Ca2+ Flux Assays-- HEK 293 cells stably expressing human receptors CCR1 and CCR5 were derived by subcloning the cDNAs into pcDNA3 and transfection using Transfectam (Promega, Southampton, UK) according to the manufacturer's methods. Stably transfected cells were selected in 0.8 mg/ml G418. To detect ligand-induced calcium ion fluxes, cells were loaded with Fura-2-AM, then, ~6 × 106 cells were incubated at 37 °C in a continuously stirred cuvette in a Perkin-Elmer LS50 Spectrometer (340 nm (lambda ex); 500 nm (lambda em)) and fluorescence emission recorded every 100 ms. After 2 min, ligand was added to a defined concentration and fluorescence recorded every 100 ms for a further 2 min. To control for day-to-day experimental variation, a full dose-response curve for LD78beta was performed each time a different ligand was tested.

HIV Entry Assays-- 5 × 104 CEMx174-CCR5 cells, pretreated with chemokine for 30 min, were incubated for 4 h with luciferase virus (5 ng of p24), pseudotyped by either JR.FL or ADA according to a previously described protocol (17). Medium was then changed and the luciferase activity measured 3 to 5 days post-infection. The extent of inhibition of HIV entry was determined by comparing luciferase activity of chemokine-treated cells, with untreated cells. For studies with the replication-competent SF162 virus, protocols were similar to those described elsewhere (30). Briefly, CEMx174-CCR5 cells, or PHA/IL-2-activated PBMC from HIV1 seronegative donors, were pretreated with chemokine then infected overnight at 37 °C with SF162 virus (m.o.i. ~0.04). The medium was then changed, and the p24 concentration subsequently determined after 5 days at 37 °C using enzyme-linked immunosorbent assay. Percentage inhibition of HIV entry was calculated relative to chemokine-untreated controls.

Statistical Methods-- Analysis of the dose-response relationship between the MIP-1alpha isoforms and other chemokines in binding, calcium flux, and HIV suppressive assays were tested by "log likelihood" methodology essentially as described previously (31).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In our recent studies of MIP-1alpha receptors (23, 24) that revealed discrepancies between the binding of murine and human MIP-1alpha , we used the LD78alpha isoform of human MIP-1alpha with the amino acids ASLAADTPT at the amino terminus (Fig. 1). The close sequence similarity between LD78alpha and murine MIP-1alpha led us to examine the possible amino acid residues that may be responsible for the discriminatory binding of these ligands. The only consistent feature present in the mCCR5 and hD6 ligands but absent in human LD78alpha is a proline residue at position 2 of the mature protein (Fig. 1). The serine residue at position 2 of the LD78alpha we have used may therefore be preventing optimal receptor interaction. The alternative human MIP-1alpha isoform, LD78beta , however, does have a proline residue in position 2, with two reciprocal serine/glycine swaps in the region between cysteines 3 and 4 being the only other differences between the LD78alpha and -beta proteins (Fig. 1) (25, 26). To simplify the nomenclature, and emphasize the serine/proline residue difference at position 2 in the two isoforms, we henceforth refer to LD78alpha and -beta as MIP-1alpha S and MIP-1alpha P, respectively.


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Fig. 1.   Alignment of the predicted protein sequences of mature beta -chemokines. Human and murine sequences are prefixed with h and m, respectively. The second residue of each protein is enlarged, enboldened, and underlined. Ability to bind to human D6 and murine CCR5 is indicated with a "+" to the right of the alignment. Non-binding chemokines are labeled with a "-." Binding information is from Refs. 23 and 24 and Graham (G. J. Graham and R. J. B. Nibbs, unpublished observations).

MIP-1alpha P Interacts with High Affinity with Both Murine and Human CCR5 and D6-- To test the importance of these isoform differences, we have now compared the ability of MIP-1alpha P and MIP-1alpha S to interact with known murine MIP-1alpha receptors stably expressed on CHO cells (Table I). In addition, we have tested the -4 presumed naturally secreted form of MIP-1alpha S (see Introduction) as well as the corresponding -4 variant of MIP-1alpha P to examine the impact of this truncation on receptor interactions. Our results demonstrate (Table I) that in contrast to MIP-1alpha S and MIP-1alpha S-4 (Kd values of 136 and 133 nM, respectively), MIP-1alpha P binds with high affinity to murine CCR5 (Kd of 1.1 nM). The importance, for this high affinity binding, of the proline residue at position 2 of MIP-1alpha P is indicated by the fact that MIP-1alpha P-4 behaves like MIP-1alpha S and MIP-1alpha S-4 on murine CCR5 (Kd 127 nM). Murine MIP-1alpha , which carries a proline residue at position 2 of the mature protein, binds to murine CCR5 in a manner similar to that seen with MIP-1alpha P, suggesting that this human variant behaves more like the murine MIP-1alpha on CCR5 than does the more common MIP-1alpha S isoform.

                              
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Table I
MIP-1alpha P shows high affinity binding to murine CCR5 and D6, but not CCR1
Dissociation constants (in nM) for displacement of 125I-mMIP-1alpha from CHO cells expressing murine MIP-1alpha receptors, by different forms of human and murine MIP-1alpha . Dissociation constants were calculated using LIGAND software (29) from data derived from displacement experiments performed as described under "Experimental Procedures."

Similarly, the presence of the proline residue at position 2 of MIP-1alpha P allowed it to bind with high affinity to murine D6 (Kd 5.5 nM) in contrast to the other three isoforms tested (Kd > 200 nM). Again the comparison with murine MIP-1alpha indicates that the MIP-1alpha P isoform binds to D6 with an affinity that more resembles murine MIP-1alpha than does any of the other human variants tested.

In contrast to the data obtained with murine CCR5 and D6, studies with murine CCR1 expressing cells indicate that the proline at position 2 is not essential for high affinity binding of the chemokines to this receptor, indeed, the Kd observed for the interaction of full-length proteins with murine CCR1 was markedly higher than that seen with the -4 truncated isoforms. Additionally, MIP-1alpha P demonstrates a substantially higher binding affinity to that seen with murine MIP-1alpha indicating that the apparent equivalence of MIP-1alpha P and muMIP-1alpha on murine CCR5 and D6 does not extend to muCCR1.

CHO cells expressing human receptors were also tested and showed similar results (Fig. 2 and Table II). Thus, with human D6, while the MIP-1alpha P isoform binds with high affinity (Kd 5.5 nM), the -4 variants of the two isoforms bind only weakly (Kd 77 nM for MIP-1alpha S-4 and Kd 124 nM for MIP-1alpha P-4) and MIP-1alpha S does not exhibit an enhanced binding affinity over its -4 variant (Fig. 2A). Again, as seen with the murine receptors, the Kd of MIP-1alpha P for D6 is more similar to that of murine MIP-1alpha than any of the other isoforms tested (Table II). The differences between MIP-1alpha S and MIP-1alpha P are, however, somewhat less stark than those seen with the murine receptor. With human CCR5, there is an approximately 4-6-fold higher binding affinity of MIP-1alpha P (Kd 6.2 nM) than is seen for the -4 MIP-1alpha proteins (Kd 25 and 36 nM), and the addition of the ASLA amino acids to MIP-1alpha S-4 results in a further reduction in binding affinity (Fig. 2B). Curiously, muMIP-1alpha binds in a manner more similar to that seen with either of the -4 variants than that seen with MIP-1alpha P. The clear requirement for the proline residue in position 2 for enhanced binding of human MIP-1alpha to human CCR5 suggests that the simple presence of the analogous residue in murine MIP-1alpha is insufficient in the context of the other evolutionary changes in this protein, to mediate high affinity binding to human CCR5. Again, in a manner similar to that observed for murine CCR1, the proline at position 2 of MIP-1alpha P appears to be unnecessary for the interaction of this ligand with human CCR1, with the full-length versions of MIP-1alpha S and MIP-1alpha P, showing a consistently lower affinity interaction with human CCR1 than either of the -4 variants (Fig. 2C). Again, in common with the data obtained using murine CCR1, murine MIP-1alpha binds more like the human -4 variants than either of the two full-length human MIP-1alpha isoforms (Table II).


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Fig. 2.   The proline residue at position 2 of MIP-1alpha P enhances affinity for human CCR5 and D6, but not CCR1. Displacement of 125I-mMIP-1alpha by unlabeled human MIP-1alpha proteins, from CHO cells expressing: A, human D6; B, human CCR5; and C, human CCR1. Experiments were performed on 105 cells incubated at room temperature in the presence of 0.4% azide for 90 min before washing with ice-cold phosphate-buffered saline. Cells were lysed in 0.1% SDS and the remaining 125I-mMIP-1alpha in the lysate counted. , MIP-1alpha P; diamond , MIP-1alpha P-4; open circle , MIP-1alpha S; Delta , MIP-1alpha S-4.

                              
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Table II
MIP-1alpha P shows high affinity binding to human CCR5 and D6 but not to CCR1
Dissociation constants (in nM) for displacement of 125I-mMIP-1alpha from CHO cells expressing human receptors, by different forms of human and murine MIP-1alpha . Dissociation constants were calculated using LIGAND software (29).

It is important to note that while all the above binding data was obtained from displacement studies utilizing radiolabeled murine MIP-1alpha and the relevant cold competitor, very similar dissociation constants have been obtained from direct binding studies using radiolabeled human MIP-1alpha isoforms.

MIP-1alpha Isoform Binding Variations Are Reflected in Signaling Potency-- To test whether the observed isoform binding variations are also reflected in the dose response for signaling through human CCR5 and CCR1, ligand-induced mobilization of Ca2+ was studied. The transfected CHO cell lines used in the above binding studies are inefficient at fluxing Ca2+ and for this reason we have used stably transfected HEK cells in the Ca2+ fluxing studies reported in this paper. Binding analyses using these cell lines indicates that the tested ligands for CCR1 and CCR5 bind with equivalent affinities to the receptors expressed in either CHO cells or HEK cells suggesting the likely equivalence of the receptors expressed on these different heterologous cells types. With full-length proteins on CCR5, half-maximal signaling potency is seen at approximately 10 nM for MIP-1alpha S and at approximately 500 pM for MIP-1alpha P (Fig. 3A). The dose-response curves obtained for these two full-length chemokines are highly significantly different (p < 0.0002) indicating, as was seen with the binding studies, that MIP-1alpha P is a markedly better ligand for CCR5 than MIP-1alpha S. The MIP-1alpha S-4 variant shows a slight, but significant (p < 0.001), increase in signaling potency through CCR5 compared with MIP-1alpha S, while removal of the terminal 4 amino acids from the MIP-1alpha P isoform significantly reduces its activity approximately 10-fold (p < 0.0002). Dose-response curves obtained using the two -4 variants were not significantly different and half-maximal activity was observed for both at concentrations of approximately 5 nM. The potency of signaling with MIP-1alpha P is significantly higher (approximately 10-fold; p < 0.001) than that seen with RANTES (half-maximal activity seen at 2.5 nM) and ~100-fold higher than that seen with MIP-1beta (p < 0.0002). MIP-1alpha P is therefore the most active natural CCR5 agonist described to date. We have detected Ca2+ fluxes with MCP2, which has recently been identified as a CCR5 ligand (32), but this ligand is not as potent as MIP-1beta in this assay (data not shown). Murine MIP-1alpha signaling potency through human CCR5 accurately reflected the binding data with a half-maximal activity being observed at 5 nM, a concentration that was indistinguishable from that seen with either of the -4 variants (data not shown).


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Fig. 3.   The proline residue at position 2 of MIP-1alpha P enhances agonist activity through CCR5, but not CCR1. A, CCR5-293 cells; B, CCR1-293 cells. Dose-response curves for ligand-induced calcium ion fluxes in HEK293 cells expressing human MIP-1alpha receptors and loaded with Fura-2. Readings were taken every 100 ms for 2 min (340 nm (lambda ex); 500 nm (lambda em)) and ligand added after ~40 s. The peak of ligand-induced fluorescence increase is presented as a percentage of the maximum achievable peak with each particular transfected cell line. , MIP-1alpha P; diamond , MIP-1alpha P-4; open circle , MIP-1alpha S; Delta , MIP-1alpha S-4; ×, hRANTES; , hMIP-1beta .

Signaling through human CCR1 again reflects the results from the binding studies and indicates no significant differences in signaling potency between the full-length isoforms of human MIP-1alpha which both display half-maximal fluxing capacity at approximately 2.5 nM (Fig. 3B). The -4 variants, however, while they are not different from each other in calcium fluxing ability, have a significantly higher signaling potency than the full-length peptides (p < 0.02) with half-maximal activity being observed at approximately 750 pM. In our hands, RANTES demonstrates a half-maximal fluxing potency at approximately 15 nM on human CCR1 and is thus significantly less potent than any of the MIP-1alpha isoforms tested (p < 0.001). Murine MIP-1alpha mediated a half-maximal flux at approximately 600 pM, again reflecting the similar binding potency of this ligand to the two -4 variants on human CCR1.

We have so far been unable to demonstrate a signaling role for human D6 (24) and, despite initial encouraging data (23), have been unable to reproducibly confirm a signaling role for murine D6. Thus the functional consequences of the high affinity binding of MIP-1alpha P to either the murine or human orthologues of this receptor remain to be determined.

In summary, these results show that the proline residue at position 2 of MIP-1alpha P is responsible for the enhanced binding of this isoform to murine and human D6 and CCR5, and for its strong activation of human CCR5. Conversely, interaction with CCR1 is lessened in the presence of APLA or ASLA at the NH2 terminus of human MIP-1alpha . These conclusions have been supported using -1, -2, and -3 forms of MIP-1alpha S with NH2 termini of SLAADTPT, LAADTPT, and AADTPT, respectively. In short, on human CCR1 and -5, and D6, the -2 and -3 forms behave like the -4 variants, while -1MIP-1alpha S acts like full-length MIP-1alpha S protein (data not shown). Truncation beyond the -4 position reduces CCR1 and -5 activation properties further (not shown).

MIP-1alpha P Is a Potent Natural Antagonist of HIV Interactions with CCR5-- Given the strong affinity of MIP-1alpha P for CCR5, we investigated the potency of this chemokine as an HIV suppressive agent. Studies were carried out to investigate the potency of full-length and -4 variants of both isoforms of human MIP-1alpha in suppressing entry of JRFL envelope pseudotyped virus into CEMx174-CCR5 cells. These studies demonstrated MIP-1alpha P to be a significantly (p < 0.002) more potent suppressor of HIV entry than either MIP-1alpha S or the -4 variants (Fig. 4A). Greater than half-maximal inhibition of JRFL pseudotyped viral entry was achieved with concentrations of MIP-1alpha P as low as 5 ng/ml while 10-20-fold higher concentrations of the other human MIP-1alpha variants were required before 50% inhibition of viral entry was achieved. Similar results were seen with virus pseudotyped with the envelope of the ADA HIV1 strain, although all the variants were less able to inhibit entry (Fig. 4B).


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Fig. 4.   MIP-1alpha P is a more potent inhibitor of HIV entry through CCR5 than other MIP-1alpha variants and RANTES. Chemokine-induced inhibition of entry into CEMx174 cells expressing human CCR5 by luciferase reporter virus pseudotyped by Envs of HIV macrophage-tropic strains JRFL and ADA (A and B), respectively, or C, by replication-competent SF162. D, chemokine-induced inhibition of entry of replication-competent SF162 virus into peripheral blood mononuclear cells stimulated with PHA and IL-2. Assays are described in more detail under "Materials and Methods." , MIP-1alpha P; diamond , MIP-1alpha P-4; open circle , MIP-1alpha S; Delta , MIP-1alpha S-4; ×, hRANTES; , AOP-RANTES.

RANTES has been reported to be a much more potent antagonist of HIV entry through CCR5 than MIP-1alpha S, MIP-1beta , and MCP2 (17-19). Therefore, further experiments were performed using replication-competent virus entry into CEMx174-CCR5 cells, and peripheral blood mononuclear cells (shown in Fig. 4, C and D, respectively), to compare MIP-1alpha P to RANTES. These studies suggest that in addition to being by far the most effective human MIP-1alpha variant in suppressing HIV entry into target cells, MIP-1alpha P is also at least as potent as RANTES. In fact in repeated experiments, MIP-1alpha P was consistently better than RANTES as an HIV suppressive chemokine, however, these differences did not reach levels of statistical significance. Again, while MIP-1alpha P was consistently less active as an HIV suppressive chemokine than the NH2-terminal modified form of RANTES, AOP-RANTES (33), this difference did not reach levels of statistical significance (Fig. 4, C and D, and see "Discussion"). Note that RANTES and MIP-1alpha S-4 can enhance HIV entry into peripheral blood mononuclear cells at the lower concentrations tested, an effect not seen with MIP-1alpha P or AOP-RANTES (Fig. 4D). In contrast to the CCR5-dependent viruses, no inhibition of CXCR4-dependent viral entry was observed using any of the MIP-1alpha isoforms tested thus confirming the CCR5 dependent selectivity of the MIP-1alpha P inhibition (data not shown). These results present MIP-1alpha P as one of the most potent naturally occurring inhibitors of HIV1 entry through CCR5.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies from our own and other laboratories have demonstrated the curious inability of human MIP-1alpha to bind with high affinity to CCR5 and D6 to which the closely related (74% identity at the amino acid level) murine MIP-1alpha protein binds strongly (23, 24, 34-36). We now reveal that the basis for this is the absence of a proline residue at position 2 of the most commonly used isoform of human MIP-1alpha (MIP-1alpha S/LD78alpha ). Our studies on the naturally occurring non-allelic variant of human MIP-1alpha (MIP-1alpha P/LD78beta ) which has a proline at position 2, reveal this to be active as a potent CCR5 and D6 ligand. It therefore more closely resembles murine MIP-1alpha than do any of the other human MIP-1alpha isoforms, suggesting that MIP-1alpha P may be a closer functional human homologue of murine MIP-1alpha than MIP-1alpha S. It appears, therefore, that despite the similar levels of identity between MIP-1alpha S and P and murine MIP-1alpha , MIP-1alpha S is a functionally evolving variant of MIP-1alpha . Our results suggest that MIP-1alpha S should be regarded as a structurally related but functionally distinct chemokine which has substantially lost its ability to interact with the CCR5 and D6 receptors but which has apparently enhanced binding to CCR1 due to the proposed truncation of MIP-1alpha S by 4 amino acids during release from the cell. Neither murine MIP-1alpha nor MIP-1alpha P are released from mammalian cells as -4 variants (20, 28, 37)4 and thus this putative alternative signal peptidase cleavage may be regarded as a component of the functional evolution of this human chemokine. The importance of the amino terminus to chemokine function is well documented (32, 38-41) and it is therefore an ideal position for evolutionary modulation of function.

A major consequence of the enhanced interactions between MIP-1alpha P and CCR5 is that this chemokine variant is now identified as being as potent as RANTES and AOP-RANTES in antagonizing CCR5-mediated cellular entry by HIV1. It is important, however, to point out that MIP-1alpha P while not reaching levels of significance, was a consistently more effective HIV suppressive agent than RANTES. This coupled with the markedly and significantly lower ability of RANTES compared with MIP-1alpha P to flux Ca2+ following CCR5 binding, may suggest that the inability to demonstrate statistical significance in HIV suppression is a limitation of the assay system used and this is currently being investigated in more detail in our laboratories. Intriguingly, and as shown in Fig. 4, A and B, while it is consistently difficult to achieve higher than 50% inhibition of entry by JRFL or ADA pseudotyped viruses using other MIP-1alpha variants, MIP-1alpha P can mediate near 100% inhibition with reasonable ease. Similar effects have been demonstrated for the synthetic NH2-terminal variant of RANTES, AOP-RANTES, with recent data suggesting this to be a consequence of alternative subcellular deposition of internalized receptors, and associated impairment of recycling of receptors (42). This may suggest that MIP-1alpha P, in contrast to MIP-1alpha S, alters receptor trafficking post-ligand binding and this is currently being investigated in our laboratory.

These results attesting to the importance of a proline residue at position 2 of MIP-1alpha are likely to have implications for our understanding of the interactions between other chemokines and their receptors. For example, it is likely that proline 2 in other beta -chemokines is necessary for high affinity binding to D6. This, however, is not sufficient for D6 interaction, as SDF1 also contains a proline residue at position 2 yet shows no potential to bind to D6 (data not shown). Thus, proline 2 must be presented in the context of a beta -chemokine to permit high affinity D6 interaction, while with CCR5 other domains are likely important in restricting the ligands for this receptor to MIP-1alpha , -1beta , RANTES, and MCP2. Interestingly, the NH2 terminus Xaa-Pro in chemokines has been demonstrated to be a target of dipeptidyl peptidase IV (CD26) (41, 43, 44). We would predict that MIP-1alpha P, but not full-length nor -4 forms of MIP-1alpha S, would be cleavable by this protease with dramatic changes in its properties, specifically a near total loss of interaction with D6, a reduced signaling capacity (and inhibition of HIV entry) through CCR5, and enhanced CCR1 activity. In fact, studies we have performed on a -2 form of MIP-1alpha S (not shown) suggest that these properties will be seen with CD26-cleaved MIP-1alpha P.

The role of the proline residue in CCR5 activation is of interest in light of recent data indicating the crucial role of proline 2 in SDF1 activation of its receptor CXCR4, which acts as an entry cofactor for T-tropic HIV1 strains. Indeed, mutation of this residue to glycine generates a high affinity CXCR4 antagonist (45). It is intriguing that both major HIV entry co-receptors have a strong preference for a proline residue at position 2 during receptor activation. Moreover, as proposed above for MIP-1alpha P, SDF1 has been demonstrated to lose its anti-HIV and chemotactic activities upon removal of the first two amino acids by CD26 cleavage (43). Targeted reduction of CD26 activity in vivo may therefore enhance the activity of these HIV suppressive chemokines.

In addition to being of general interest to workers in the chemokine and HIV fields, our data also open up a number of potential therapeutic avenues of research. For example, earlier studies have demonstrated that the MIP-1alpha P copy number varies between individuals, and this gene can in fact be absent from some individuals (25, 26). beta -Chemokine production has been reported to be associated with a number of inflammatory and autoimmune diseases (1, 2), and deletion of the MIP-1alpha gene in mice dramatically alters responses to several infectious agents (22). In addition, beta -chemokine production is clearly of importance in regulating the pathogenesis of AIDS (6, 12-16). Thus, given the ease with which MIP-1alpha P is transcribed and translated (21, 26, 28)5 MIP-1alpha P gene dosage may alter an individuals' response in these pathological situations. In particular, in light of the potent HIV entry inhibition by MIP-1alpha P described here, it would be of interest to test whether gene copy number affects the rate of progression to AIDS in HIV-infected individuals.

The demonstration of the potent HIV suppressive action of MIP-1alpha P also indicates that it is possible that, as has been demonstrated with the chemokine RANTES (33, 39), amino-terminal variants of this protein, such as AOP-linked MIP-1alpha P, may exhibit enhanced HIV1 entry inhibition and/or receptor antagonism and have potential as HIV1 therapeutics.

    ACKNOWLEDGEMENTS

We thank Prof. John Wyke and Dr. Paul Clapham for critical reading of the manuscript. R. J. B. N. thanks Dr. Amanda Wilson for manuscript review and support services.

    FOOTNOTES

* This work was supported by the Cancer Research Campaign (to G. J. G), National Institutes of Health Grants CA72149, AI36057, and AI1384 (to N. R. L.), and AmFar Grant 02580-23-RGV (to J. Y. and N. R. L).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Elizabeth Glaser Scholar of the Pediatric AIDS Foundation.

** To whom correspondence should be addressed. Tel.: 44-141-330-3982; Fax: 44-141-942-6521; E-mail: g.graham{at}beatson.gla.ac.uk.

2 R. J. B. Nibbs, unpublished data.

3 A. N. Parker, G. J. Graham, A. R. Sim, S. C. Clark, and I. B. Pragnell, manuscript in preparation.

4 G. J. Graham, unpublished observations.

5 G. J. Graham and R. J. B. Nibbs, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; MCP2, monocyte chemotactic protein-2; MIP-1alpha and -beta , macrophage inflammatory protein-1alpha and -beta ; CHO, Chinese hamster ovary.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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