Mechanism of Transdominant Inhibition of CCR5-mediated HIV-1 Infection by ccr5Δ32

Human chemokine receptor 5 (CCR5) functions as a co-receptor for Human immunodeficiency virus (HIV-1) infection. CCR5 is a seven-transmembrane cell surface receptor. Recently, a naturally occurring mutation of CCR5, ccr5Δ32, has been described. A small number of Caucasians are homozygouslyccr5Δ32/ccr5Δ32, while a larger number of individuals are heterozygously CCR5/ccr5Δ32. Theccr5Δ32/ccr5Δ32 genotype has been linked to a phenotype that is “highly” protected from HIV-1 infection. On the other hand, several studies have shown that the CCR5/ccr5Δ32 genotype confers “relative” protection from AIDS with onset of disease being delayed by 2–4 years. Although it is known that peripheral blood lymphocytes from heterozygous individuals (CCR5/ccr5Δ32) support ex vivo HIV-1 replication at a reduced level compared with CCR5/CCR5 cells, the molecular basis for this observation is unknown. Here we report on events that post-translationally modify CCR5. We show that CCR5 progresses through the endoplasmic reticulum prior to appearing on the cell surface. Mature CCR5 can be post-translationally modified by phosphorylation and/or co-translationally by multimerization. By contrast, mutant ccr5Δ32, although retaining the capacity for multimerization, was incapable of being phosphorylated. ccr5Δ32 heterocomplexes with CCR5, and this interaction retains CCR5 in the endoplasmic reticulum resulting in reduced cell surface expression. Thus, co-expression in cells of ccr5Δ32 with CCR5 produces a trans-inhibition by the former of ability by the latter to support HIV-1 infection. Taken together, our findings suggest CCR5/ccr5Δ32 heterodimerization as a molecular explanation for the delayed onset of AIDS inCCR5/ccr5Δ32 individuals. Human chemokine receptor 5 (CCR5) functions as a co-receptor for Human immunodeficiency virus (HIV-1) infection. CCR5 is a seven-transmembrane cell surface receptor. Recently, a naturally occurring mutation of CCR5, ccr5Δ32, has been described. A small number of Caucasians are homozygouslyccr5Δ32/ccr5Δ32, while a larger number of individuals are heterozygously CCR5/ccr5Δ32. Theccr5Δ32/ccr5Δ32 genotype has been linked to a phenotype that is “highly” protected from HIV-1 infection. On the other hand, several studies have shown that the CCR5/ccr5Δ32 genotype confers “relative” protection from AIDS with onset of disease being delayed by 2–4 years. Although it is known that peripheral blood lymphocytes from heterozygous individuals (CCR5/ccr5Δ32) support ex vivo HIV-1 replication at a reduced level compared with CCR5/CCR5 cells, the molecular basis for this observation is unknown. Here we report on events that post-translationally modify CCR5. We show that CCR5 progresses through the endoplasmic reticulum prior to appearing on the cell surface. Mature CCR5 can be post-translationally modified by phosphorylation and/or co-translationally by multimerization. By contrast, mutant ccr5Δ32, although retaining the capacity for multimerization, was incapable of being phosphorylated. ccr5Δ32 heterocomplexes with CCR5, and this interaction retains CCR5 in the endoplasmic reticulum resulting in reduced cell surface expression. Thus, co-expression in cells of ccr5Δ32 with CCR5 produces a trans-inhibition by the former of ability by the latter to support HIV-1 infection. Taken together, our findings suggest CCR5/ccr5Δ32 heterodimerization as a molecular explanation for the delayed onset of AIDS inCCR5/ccr5Δ32 individuals. Human immunodeficiency virus (HIV-1) 1The abbreviations used are: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; aa, amino acid(s); HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PHA, phytohemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin. uses CD4 as the primary receptor and chemokine co-receptors to enter target cells (1Clapham P.R. Weiss R.A. Nature. 1997; 388: 230-231Crossref PubMed Scopus (83) Google Scholar). Chemokine receptors belong to the superfamily of G protein-coupled receptors that have seven transmembrane domains. Chemokines are a family of small proteins (7–16 kDa) that can be operationally divided in two subgroups. The α subfamily (CXC) is distinguished from the β subfamily (CC) by the insertion of a single amino acid between the first and the second cysteine residues. The binding of chemokines to their receptors induces a rapid calcium influx and inflammatory responses (2Murphy P.M. Cytokine Growth Factor Rev. 1996; 7: 47-64Crossref PubMed Scopus (283) Google Scholar). The CXC chemokine receptor for stromal cell-derived factor-1 designated CXCR4 (3Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1755) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clarck-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1484) Google Scholar) was initially shown to be a co-receptor for T-cell-tropic (T-tropic) HIV-1s (5Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3643) Google Scholar). Based on the finding that chemokines (CC-β) RANTES, MIP-1α, and MIP-1β inhibit infection by macrophage-tropic (M-tropic) HIV-1 isolates (6Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2644) Google Scholar), subsequent studies revealed that CCR5 functions as a major co-receptor for M-tropic viruses (7Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2452) Google Scholar, 8Choe H. Farzan M. Sun Y. Sullivan N. Rollins B. Ponath P.D. Wu L. Mackay C.R. LaRosa G. Newman W. Gerard N. Gerard C. Sodroski J. Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2095) Google Scholar, 9Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M. Marzio P.D. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3206) Google Scholar, 10Doranz B.J. Rucker J. Yi Y. Smyth R.J. Samson M. Peiper S.C. Parmentier M. Collman R.G. Doms R.W. Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 11Dragic T. Litwin V. Allaway G.P. Martin S.R. Huang Y. Nagashima K.A. Cayanan C. Madon P.J. Koup R.A. Moore J.P. Paxton W.A. Nature. 1996; 381: 667-673Crossref PubMed Scopus (2821) Google Scholar). Accordingly, further investigations have demonstrated the existence, in some ethnic groups (e.g. Caucasians), of a natural genetic mutation inCCR5 (ccr5Δ32, an internal 32-nucleotide deletion in the CCR5 open reading frame). Homozygousccr5Δ32/ccr5Δ32 genotype confers resistance to HIV-1 infection in vitro and in vivo (12Liu R. Paxton W.A. Choe S. Ceradini D. Martin S.R. Horuk R. MacDonald M.E. Stuhlmann H. Koup R.A. Landau N.R. Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar). However, the incidence of homozygocity in Caucasians is low (1%), while heterozygous (CCR5/ccr5Δ32) individuals exist more prevalently (up to 20% in some populations). There is evidence that CCR5/ccr5Δ32 heterozygotes progress more slowly to AIDS (13Dean M. Carrington M. Winkler C. Huttley G.A. Smith M.W. Allikmets R. Goedert J.J. Buchbinder S.P. Vittinghoff E. Gomperts E. Donfield S. Vlahov D. Kaslow R. Saah A. Rinaldo C. Detels R. HGDS. MACS. MHCS. SFCC. O'Brien S.J. Science. 1996; 273: 1856-1862Crossref PubMed Scopus (2196) Google Scholar, 14Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtomboy G. Georges M. Imai T. Rana S. Yi Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2473) Google Scholar, 15Huang Y.X. Paxton W.A. Wolinsky S.M. Neumann A.U. Zhang L.Q. He T. Kang S. Ceradini D. Jin Z. Yazdanbakhsh K. Kunstman K. Erickson D. Dragon E. Landau N.R. Phair J. Ho D.D. Koup R.A. Nat. Med. 1996; 2: 1240-1243Crossref PubMed Scopus (1215) Google Scholar, 16Michael N.L. Chang G. Louie L.G. Mascola J.R. Dondero D. Birx D.L. Sheppard H.W. Nat. Med. 1997; 3: 338-340Crossref PubMed Scopus (415) Google Scholar). Currently, how heterozygocity (CCR5/ccr5Δ32) mechanistically impacts disease progression is unknown. It has, however, been observed thatCCR5/ccr5Δ32 PBMCs are less infectible in vitroby M-tropic HIV-1s than CCR5/CCR5 cells (12Liu R. Paxton W.A. Choe S. Ceradini D. Martin S.R. Horuk R. MacDonald M.E. Stuhlmann H. Koup R.A. Landau N.R. Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar). Although the level of cell surface expression of CCR5 in the uninfected population is quite heterogeneous, varying up to 20-fold between individuals (17Moore J.P. Science. 1997; 276: 51-52Crossref PubMed Scopus (219) Google Scholar), one study has found that CCR5/ccr5Δ32 T-cells are markedly reduced for surface expression of CCR5 compared withCCR5/CCR5 counterparts (18Wu B.L. Paxton W.A. Kassam N. Ruffing N. Rottman J.B. Sullivan N. Choe H. Sodroski J. Newman W. Koup R.A. Mackay C.R. J. Exp. Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar). Accordingly, a correlation between surface CCR5 expression and infectibility by M-tropic HIV-1s is suggested (18Wu B.L. Paxton W.A. Kassam N. Ruffing N. Rottman J.B. Sullivan N. Choe H. Sodroski J. Newman W. Koup R.A. Mackay C.R. J. Exp. Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar). The mechanism through which ccr5Δ32 might affect CCR5-function remains to be clarified. To assess this issue we studied the effect of ccr5Δ32 on the processing, stability, and cell surface expression of CCR5. We found that 1) CCR5 is post-translationally phosphorylated upon MIP-1β stimulation of cells; 2) intracellularly, CCR5 exists as either CCR5/CCR5 or CCR5/ccr5Δ32 multimers; 3) CCR5 and ccr5Δ32 are found in different cellular locales (the former is predominantly on the cell surface while the latter is retained in the ER); and 4) co-expression of ccr5Δ32 inhibited surface expression of CCR5 and CCR5-mediated infection by M-tropic HIV-1 isolates. All constructs were derived from pCCR5 (5Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3643) Google Scholar). CCR5 mutants, cloned into pcDNA3 (Invitrogen), include pCMV/CCR5Δcyt (aa 1–303), pCMV/CCR5/6TM (aa 1–279), pCMV/CCR5/5TM (aa 1–235), and pCMV/CCR5Δ32 (aa 1–187). Each mutant was generated by polymerase chain reaction with the HA epitope fused to each cDNA at the 3′ terminus. pCMVCCR5-Flag was a gift from Ron Willey (National Institutes of Health). HeLa cells were propagated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. PBMCs were activated for 3 days in RPMI with 10% fetal bovine serum containing PHA and then washed and resuspended into the same medium without PHA and with 100 units/ml interleukin-2 (Boehringer Mannheim). PBMCs were exposed to M-tropic HIV-1 isolate AD8 (500 units of TCID50) for 1 h at 37 °C, washed, and then resuspended into fresh medium. Virus replication was monitored by reverse transcriptase assay as described previously (19Huang L. Joshi A. Willey R. Ornstein J. Jeang K.-T. EMBO. J. 1994; 13: 2886-2896Crossref PubMed Scopus (86) Google Scholar). Transfection of HeLa cells was performed using calcium phosphate. 24 h after transfection, cells were washed twice in DMEM without methionine and cysteine starved for 30 min in the same medium at 37 °C. 35SMethionine + cysteine (translabel ICN) at 1 mCi/ml final concentration was added to the cells. The cells were then incubated at 37 °C for the indicated amount of pulse time and then washed and resuspended in DMEM with methionine and cysteine for the indicated chase times. For immunoprecipitation, identical protein amounts were suspended into 1 ml of Triton lysis buffer (0.5% Triton X-100, 300 mm NaCl, 50 mm Tris, pH 7.4, 0.2 mm phenylmethylsulfonyl fluoride) and incubated for 2 h at 4 °C with either anti-Flag M2 (Eastman Kodak Co.) or anti-HA (12CA5, Boehringer Mannheim). A mixture of protein A- and protein G-Sepharose (Pharmacia Biotech Inc.) was added to each sample followed by a 1-h incubation at 4 °C. Three washes were performed in Triton wash buffer (0.1% Triton X-100, 300 mm NaCl, 50 mm Tris, pH 7.4, 0.2 mm phenylmethylsulfonyl fluoride) and a final wash in SDS/deoxycholate buffer (300 mm NaCl, 50 mm Tris, pH 7.4, 0.1% SDS, 0.1% deoxycholate). The immunoprecipitated products were solubilized in 1 × loading buffer (125 mm Tris, pH 6.8, 20% glycerol, 2% SDS, 2% β-mercaptoethanol, 0.01% bromphenol blue) and resolved by SDS-PAGE. HeLa cells were seeded onto coverslips and transfected. 24 h later, cells were fixed with fresh 4% paraformaldehyde, pH 7.0, for 10 min at room temperature. Fixed cells were permeabilized with a 2-min wash in 100% methanol at room temperature followed by several washes in PBS with 4% bovine serum albumin (PBS/BSA). Appropriately diluted primary antibody was incubated with coverslips overnight at 4 °C. Excess antibody was removed with four washes in PBS/BSA. Species-specific second antibody conjugated to Texas Red (Cappel) was then reacted with the coverslips for 1 h at room temperature followed with four washes in PBS/BSA. The final samples were mounted onto slides and visualized using a Ziess Axiophot confocal microscope. Two-hybrid assays were performed according to manufacturer's protocols (CLONTECH). To ask whether CCR5 forms an oligomer, HeLa cells were transfected with pCMV/CCR5-Flag, which contains the Flag epitope fused to the C terminus of CCR5. 24 h later, cells were pulsed with 35Smethionine + cysteine for 15 min, washed, and then chased in complete medium for 1 h. Extracts prepared from pulsed and pulsed + chased cells were immunoprecipitated using anti-Flag M2. The immunoprecipitates were resuspended into either 1 × loading buffer (Fig. 1 A, lanes 1 and2) or “native” loading buffer without β-mercaptoethanol with a reduced amount of SDS (0.25%) (Fig.1 A, lanes 3 and 4). The samples were resolved by SDS-PAGE. The reduced mobility of CCR5 in the pulsed + chased sample (Fig. 1 A, lane 2) when compared with the pulsed sample (Fig.1 A, lane 1) is consistent with a post-translational modification. 2R. Willey, unpublished observation. When the same analysis was repeated using native loading buffer (Fig. 1 A, lanes 3 and 4), the mobility difference observed for pulsed (lane 3) and pulsed + chased (lane 4) samples was replicated. The native buffer-PAGE revealed additional CCR5-specific bands with sizes consistent with dimeric moieties (d; Fig.1 A, lanes 3 and 4). To characterize better multimerization potentials, we analyzed CCR5 using the yeast two-hybrid approach (TableI). In this analysis, C-terminal truncation mutants of CCR5 were found to interact with wild type CCR5. However, a mutant deleted in the first 58 amino acids failed to interact with intact CCR5. Thus, in yeasts, the CCR5-CCR5 interactive domain resides in the N-terminal portion of the protein, which encompasses the first transmembrane region.Table IYeast two-hybrid analysis of CCR5/CCR5 interactionCCR5 (1–352)TaxCCR5 (1–352)+++−CCR5Δcyt (1–303)+++−CCR5/6TM (1–279)+++−CCR5/5TM (1–235)+++−CCR5/4TM (ccr5Δ32) (1–187)+++−CCR5Δnt (58–352)−−Tax−++++Yeast were transformed by PIDCCR5wt and the different mutants using LiAc method of Gietz D. A. R.A. PubMed Scopus Google Scholar). constructs was used as interaction Jeang K.-T. 1997; PubMed Scopus Google Scholar) a A assay was performed using as as were 1 h. in a were transformed by PIDCCR5wt and the different mutants using LiAc method of Gietz D. A. R.A. PubMed Scopus Google Scholar). constructs was used as interaction Jeang K.-T. 1997; PubMed Scopus Google Scholar) a A assay was performed using as as were 1 h. The of were further We CCR5 and ccr5Δ32 in HeLa cells and for cell by an M-tropic HIV-1 T. G. A. Martin AIDS 1996; PubMed Scopus Google Scholar). A of protein is that ccr5Δ32 be a of CCR5 We found that of HeLa with wild type CCR5 as HeLa cells to infection A, lane with of CCR5 with the affect infection (Fig. 2 A, lane However, co-expression of CCR5 with either or or (ccr5Δ32) or mutants deleted for or transmembrane reduced the ability of cells to support effect was when were (Fig. 2 A, lanes M-tropic of CCR5 in of and CCR5 in cells can be as an approach in vivo infection. To a more we PBMCs from and two CCR5/ccr5Δ32 individuals. (Fig. 2 are consistent with the findings (Fig. 2 and previously (12Liu R. Paxton W.A. Choe S. Ceradini D. Martin S.R. Horuk R. MacDonald M.E. Stuhlmann H. Koup R.A. Landau N.R. Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar), that of ccr5Δ32 with CCR5 in the same cell to infection by M-tropic The infection to how ccr5Δ32 might affect vivo of CCR5. To this we used confocal to of ccr5Δ32 on CCR5. with CCR5 and ccr5Δ32 were found in different CCR5 was found on cell ccr5Δ32 in the (Fig. 3 was observed for mutants and which was found on the cell surface When CCR5 and ccr5Δ32 were in the same reduced surface of the former was The findings are consistent with CCR5-CCR5 The evidence by mobility in (Fig. lanes 1 and 2) be by other events as the C terminus of CCR5 is in and which are for G protein-coupled and phosphorylation receptor we for this transfected HeLa cells with and the cells in with either 35Smethionine + cysteine A, lanes or (Fig. 4 A, lanes for 2 h in the (Fig. 4 A, lanes or (Fig. 4 A, lanes of 100 of for were then immunoprecipitated with anti-Flag In the the amount of CCR5 was found to be between and cells with MIP-1β (Fig. 4 A, lane 1 with lane 2 and lane 3 with lane 4). However, in the same a difference in CCR5 in the of MIP-1β was observed (Fig. 4 A, 5 with lane and lane suggest that MIP-1β affect the expression of CCR5, affect the phosphorylation of this The of CCR5 is in To the of this portion of the protein to HeLa cells were transfected with a of CCR5 the fused at C terminus to a HA In this the was performed as that described in the to A that anti-HA was used for shown in 4 amounts of was found in and cells with MIP-1β lane 1 with lane However, we failed to (Fig. 4 lanes 3 and 4). suggest that an intact of CCR5 is for In our study 1) CCR5 exists as a 2) expression of ccr5Δ32 cell surface expression of wild type and 3) MIP-1β induces the phosphorylation of CCR5 in Taken together, suggest that are post-translational events for CCR5. to the observation of receptor multimerization, it is that CCR5 mutants as ccr5Δ32 are in some of post-translational that in the However, mutants, an ability to with wild type CCR5 and through this retains the wild type into the We that this type mechanism in PBMCs from CCR5/ccr5Δ32 individuals have of cell surface CCR5 when compared with CCR5/CCR5 counterparts (18Wu B.L. Paxton W.A. Kassam N. Ruffing N. Rottman J.B. Sullivan N. Choe H. Sodroski J. Newman W. Koup R.A. Mackay C.R. J. Exp. Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar) and individuals have delayed progression to disease (13Dean M. Carrington M. Winkler C. Huttley G.A. Smith M.W. Allikmets R. Goedert J.J. Buchbinder S.P. Vittinghoff E. Gomperts E. Donfield S. Vlahov D. Kaslow R. Saah A. Rinaldo C. Detels R. HGDS. MACS. MHCS. SFCC. O'Brien S.J. Science. 1996; 273: 1856-1862Crossref PubMed Scopus (2196) Google Scholar, 14Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtomboy G. Georges M. Imai T. Rana S. Yi Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2473) Google Scholar, 15Huang Y.X. Paxton W.A. Wolinsky S.M. Neumann A.U. Zhang L.Q. He T. Kang S. Ceradini D. Jin Z. Yazdanbakhsh K. Kunstman K. Erickson D. Dragon E. Landau N.R. Phair J. Ho D.D. Koup R.A. Nat. Med. 1996; 2: 1240-1243Crossref PubMed Scopus (1215) Google Scholar, 16Michael N.L. Chang G. Louie L.G. Mascola J.R. Dondero D. Birx D.L. Sheppard H.W. Nat. Med. 1997; 3: 338-340Crossref PubMed Scopus (415) Google Scholar). The of phosphorylation on CCR5 is less this a in the MIP-1β from the cell surface into the Recently, A. S. Schwartz O. J. M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; PubMed Scopus Google Scholar) that of chemokine receptors and by their to of HIV-1 the of CXCR4 is for of CXCR4 A. S. Schwartz O. J. M. Loetscher P. Baggiolini M. Virelizier J.L. Arenzana-Seisdedos F. J. Exp. Med. 1997; PubMed Scopus Google Scholar). phosphorylation of chemokine receptors is unknown. is that CCR5 phosphorylation can that HIV-1 infection. MIP-1α, and have been shown to have on HIV-1 replication in H. B. M. Nature. 1996; 382: PubMed Scopus Google Scholar). We that exists a of co-receptors for HIV-1 different have been in P.R. Weiss R.A. Nature. 1997; 388: 230-231Crossref PubMed Scopus (83) Google Scholar), that of HIV-1 infection be A. D. G. S. L. M. M. P.R. J. 1997; PubMed Google Scholar). We that a effect to a mutation has been described M.W. M. Carrington M. Winkler C. Huttley G.A. Goedert J.J. O'Brien Kaslow R. Buchbinder S. Vittinghoff E. Vlahov R. Buchbinder S. Vittinghoff E. D. K. M.W. HGDS. MACS. MHCS. SFCC. O'Brien S.J. Science. 1997; PubMed Scopus Google Scholar). the mutation with the CCR5 mutation (e.g. multimerization or remains to be However, the natural of chemokine receptor mutants might be a that resistance of for AIDS and that might for We E. Berger for gift of CCR5 and R. Willey for gift of and for