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

  • Monsef Benkirane
    Correspondence
    To whom correspondence should be addressed: LMM/NIAID/NIH, Bldg. 4, Rm. 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Tel.: 301-496-6680; Fax: 301-402-0226
    Affiliations
    Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460

    Centre de Recherche de Biochimie Macromoleculaire CNRS, ERS0155 Montpellier, France
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  • Dong-Yan Jin
    Affiliations
    Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
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  • Rene F. Chun
    Affiliations
    Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
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  • Richard A. Koup
    Footnotes
    Affiliations
    Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • Kuan-Teh Jeang
    Footnotes
    Affiliations
    Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
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  • Author Footnotes
    * This work was supported in part by funds from the AIDS Targeted Antiviral Program from the Office of the Director, National Institutes of Health (to K.-T. J.) and by National Institutes of Health Grants AI35522 and AI42397 (to R. A. K.).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 Scientist of the Pediatric AIDS Foundation.
Open AccessPublished:December 05, 1997DOI:https://doi.org/10.1074/jbc.272.49.30603
      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)
      The 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.
      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 (
      • Clapham P.R.
      • Weiss R.A.
      ). 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 (
      • Murphy P.M.
      ). The CXC chemokine receptor for stromal cell-derived factor-1 designated CXCR4 (
      • Bleul C.C.
      • Farzan M.
      • Choe H.
      • Parolin C.
      • Clark-Lewis I.
      • Sodroski J.
      • Springer T.A.
      ,
      • Oberlin 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.
      ) was initially shown to be a co-receptor for T-cell-tropic (T-tropic) HIV-1s (
      • Feng Y.
      • Broder C.C.
      • Kennedy P.E.
      • Berger E.A.
      ). Based on the finding that chemokines (CC-β) RANTES, MIP-1α, and MIP-1β inhibit infection by macrophage-tropic (M-tropic) HIV-1 isolates (
      • Cocchi F.
      • DeVico A.L.
      • Garzino-Demo A.
      • Arya S.K.
      • Gallo R.C.
      • Lusso P.
      ), subsequent studies revealed that CCR5 functions as a major co-receptor for M-tropic viruses (
      • Alkhatib G.
      • Combadiere C.
      • Broder C.C.
      • Feng Y.
      • Kennedy P.E.
      • Murphy P.M.
      • Berger E.A.
      ,
      • Choe 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.
      ,
      • Deng 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.
      ,
      • Doranz B.J.
      • Rucker J.
      • Yi Y.
      • Smyth R.J.
      • Samson M.
      • Peiper S.C.
      • Parmentier M.
      • Collman R.G.
      • Doms R.W.
      ,
      • Dragic 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.
      ). 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 (
      • Liu R.
      • Paxton W.A.
      • Choe S.
      • Ceradini D.
      • Martin S.R.
      • Horuk R.
      • MacDonald M.E.
      • Stuhlmann H.
      • Koup R.A.
      • Landau N.R.
      ). 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 (
      • Dean 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.
      ,
      • Samson 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.
      ,
      • Huang 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.
      ,
      • Michael N.L.
      • Chang G.
      • Louie L.G.
      • Mascola J.R.
      • Dondero D.
      • Birx D.L.
      • Sheppard H.W.
      ). 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 (
      • Liu R.
      • Paxton W.A.
      • Choe S.
      • Ceradini D.
      • Martin S.R.
      • Horuk R.
      • MacDonald M.E.
      • Stuhlmann H.
      • Koup R.A.
      • Landau N.R.
      ). Although the level of cell surface expression of CCR5 in the uninfected population is quite heterogeneous, varying up to 20-fold between individuals (
      • Moore J.P.
      ), one study has found that CCR5/ccr5Δ32 T-cells are markedly reduced for surface expression of CCR5 compared withCCR5/CCR5 counterparts (
      • Wu 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.
      ). Accordingly, a correlation between surface CCR5 expression and infectibility by M-tropic HIV-1s is suggested (
      • Wu 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.
      ).
      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.

      EXPERIMENTAL PROCEDURES

      Plasmid Constructs

      All constructs were derived from pCCR5 (
      • Feng Y.
      • Broder C.C.
      • Kennedy P.E.
      • Berger E.A.
      ). 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).

      Cell Culture, Infection, and Transfection

      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 (
      • Huang L.
      • Joshi A.
      • Willey R.
      • Ornstein J.
      • Jeang K.-T.
      ). Transfection of HeLa cells was performed using calcium phosphate.

      Pulse-Chase Analysis

      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. [35S]Methionine + 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.

      Protein Analysis

      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.

      Confocal Microscopy

      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.

      Yeast Two-hybrid Assay

      Two-hybrid assays were performed according to manufacturer's protocols (CLONTECH).

      RESULTS AND DISCUSSION

      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 [35S]methionine + 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.
      R. 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).
      Figure thumbnail gr1
      Figure 1HeLa cells were transfected with pCMV/CCR5-Flag. 24 h later, cells were pulse-labeled with [35S]methionine/cysteine (1 mCi/ml) for 15 min (lanes 1 and 3) and then chased for 1 h (lanes 2 and 4). Extracts from pulsed and chased cells were immunoprecipitated using anti-Flag M2 (Kodak). The immunoprecipitates were resuspended into either 1 × loading buffer (62.5 mm Tris, pH 6.8, 10% glycerol, 1% SDS, 1% β-mercaptoethanol; left panel) or native loading buffer without β-mercaptoethanol and with 0.25% SDS (right panel). Samples were resolved by 10% SDS-PAGE. Positions of monomeric (m) and dimeric (d) forms are indicated.
      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 interaction
      CCR5 (1–352)Tax
      CCR5 (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 et al. (
      • Gietz D.
      • St. Jean A.
      • Woods R.A.
      • Schiestl R.H.
      ). Tax/CCR5 constructs was used as negative control. Tax/Tax interaction (
      • Jin D.Y.
      • Jeang K.-T.
      ) represents a positive control. A qualitative colony-lift filter assay was performed using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as substrate. Strongly positive colonies (++++) appeared as dark blue within 20 min. Positives (+++) were blue within 1 h.
      The biological implications of dimerization were further examined. We co-expressed CCR5 and ccr5Δ32 in HeLa CD4+/LTR-βgal cells and assayed for cell fusion engendered by an M-tropic HIV-1 isolate, HIV-1AD8 (
      • Theodore T.
      • England G.
      • Buckler-White A.
      • Buckler C.E.
      • Martin M.A.
      • Peden K.W.
      ). A prediction of protein dimerization is that ccr5Δ32 could be a dominant negative inhibitor of CCR5 function. We found that transfection of HeLa CD4+ LTR-CAT with wild type CCR5 (Fig.2 A) did render, as expected, HeLa cells susceptible to HIV-1AD8 infection (Fig.2 A, compare lane 8 with 9). Co-transfection of CCR5 with ccr5Δcyt (CCR5 lacking the cytoplasmic tail) did not affect HIV-1AD8 infection (Fig. 2 A, lane 10). However, co-expression of CCR5 with either ccr5/6TM, or ccr5/5TM, or ccr5/4TM (ccr5Δ32) or ccr5/3TM (i.e.C-terminal mutants deleted for six, five, four, or three transmembrane regions, respectively) dramatically reduced the ability of cells to support HIV-1AD8-induced fusion. No effect was seen when pNL4–3 (T-tropic HIV-1) infections were conducted under parallel conditions (Fig. 2 A, lanes 1–7), confirming M-tropic specificity of CCR5 in these experiments. Co-expression of ccrΔ32 and CCR5 in monolayer cells can be regarded as an artificial approach modeling in vivo infection. To construct a more physiological test, we conducted infections comparing PBMCs from oneCCR5/CCR5 and two CCR5/ccr5Δ32 individuals. These results (Fig. 2 B) are consistent with the above findings (Fig. 2 A) and previously published results (
      • Liu R.
      • Paxton W.A.
      • Choe S.
      • Ceradini D.
      • Martin S.R.
      • Horuk R.
      • MacDonald M.E.
      • Stuhlmann H.
      • Koup R.A.
      • Landau N.R.
      ), confirming that simultaneous presence of ccr5Δ32 with CCR5 in the same cell reduces susceptibility to infection by M-tropic HIV-1.
      Figure thumbnail gr2
      Figure 2A, HeLa CD4+/LTR-CAT cells were transfected with pCMV/CCR5-Flag (2.5 μg) or co-transfected with pCMV/CCR5-Flag and pCMV/ccr5Δcyt or pCMV/ccr5/6TM (6TM) or pCMV/ccrR5/5TM (5TM) or pCMV/ccr5/4TM (4TM) or pCMV/ccr5/3TM (3TM) at molar ratio of 1:3. 24 h after transfection, cells were infected with either pNL4–3 (T-tropic HIV-1;left) or AD8 (M-tropic HIV-1; right). Infection and staining of cells were performed as described (
      • Kimpton J.
      • Emerman M.
      ). B, PBMCs from one homozygotic CCR5/CCR5 (+/+) individual and PBMCs from two heterozygotics CCR5/ccr5Δ32 (±) individuals were PHA stimulated for 3 days and then exposed to M-tropic isolate, HIV-1AD8 (500 units of TCID50). Virus replication was serially monitored by assaying supernatant RT every 3 days.
      The infection results led us to consider how ccr5Δ32 might affect thein vivo presentation of CCR5. To address this question, we used confocal immunofluorescence to visualize potential intracellular influences of ccr5Δ32 on CCR5. Upon staining with specific antiserum, steady-state CCR5 and ccr5Δ32 were found in distinctly different subcellular locales. While CCR5 was found on cell surfaces (Fig.3 A), ccr5Δ32 stained in the ER (Fig. 3 B). ER localization was observed for all ccr5 mutants (ccr5/6TM, ccr5/5TM, ccr5/4TM, and ccr5/3TM), except ccr5Δcyt, which was found on the cell surface (data not shown). When CCR5 and ccr5Δ32 were co-expressed in the same cell, significantly reduced surface staining of the former was seen (Fig.3 C).
      Figure thumbnail gr3
      Figure 3HeLa cells were transfected separately with pCMV/CCR5-Flag (A), pCMV/ccr5Δ32-HA (B), or co-transfected with pCMV/CCR5-Flag and pCMV/ccr5Δ32-HA at molar ratio of 1:3, respectively (C). Images in A andC are stainings using anti-Flag monoclonal antibody (Kodak).B is a staining using anti-HA monoclonal antibody (Boehringer Mannheim). Note the cell surface staining in Aand the ER staining in B and C. Fluorescent images are presented at the top, and light fields are at thebottom.
      The above findings are consistent with CCR5-CCR5 dimers. The biochemical evidence provided by mobility shifts in PAGE (Fig. 1, compare lanes 1 and 2) could also be explained by other modifying events such as phosphorylation. Because the C terminus of CCR5 is rich in serine and threonine residues, which are potential substrate sites for G protein-coupled receptor-kinase(s), and because phosphorylation could conceivably influence overall receptor function, we queried for this possibility. We, thus, transfected HeLa cells with pCMV/CCR5-Flag and labeled the cells in parallel with either [35S]methionine + cysteine (Fig.4 A, lanes 1–4) or [32P]orthophosphate (Fig. 4 A, lanes 5–8) for 2 h in the absence (Fig. 4 A, lanes 1, 3, 5, and7) or presence (Fig. 4 A, lanes 2, 4, 6, and8) of 100 ng/ml of MIP-1β. Cell extracts normalized for radioactivity were then immunoprecipitated with anti-Flag antibody. In the immunoprecipitates, the amount of 35S-labeled CCR5 was found to be equivalent between mock-treated and cells treated with MIP-1β (Fig. 4 A, compare lane 1 with lane 2 and lane 3 with lane 4). However, in the same experiment, a large difference in 32P-labeled CCR5 in the presence of MIP-1β was observed (Fig. 4 A, comparelane 5 with lane 6 and lane 7 withlane 8). These results suggest that MIP-1β does not affect the ambient expression of CCR5, but does affect the phosphorylation state of this protein.
      Figure thumbnail gr4
      Figure 4A and B, HeLa cells were transfected with either pCMV/CCR5-Flag (A) or pCMV/CCR5Δcyt-HA (B). 24 h after transfection cells were labeled with either [35S]methionine + cysteine (A, lanes 1–4, and B, lanes 1 and 2) or [32P]orthophosphate (A, lanes 5–8, andC, lanes 3 and 4) in the absence (A, lanes 1, 3, 5, and 7, and B, lanes 1 and3) or in the presence of 100 ng/ml of MIP-1β (A, lanes 2, 4, 6, and 8, and B, lanes 2 and4). Cell extracts were prepared and immunoprecipitated using anti-Flag antibody (A) or anti-HA antibody (B).
      The cytoplasmic tail of CCR5 is rich in potential phosphate acceptor sites. To examine the contribution of this portion of the protein to phosphorylation, HeLa cells were transfected with pCMV/CCR5Δcyt. This construction encodes a truncated form of CCR5 lacking the cytoplasmic tail fused at its C terminus to a HA epitope. In this instance, the experiment was performed as that described in the legend to Fig.4 A except that anti-HA was used for immunoprecipitation. As shown in Fig. 4 B, similar amounts of 35S-labeled CCR5Δcyt was found in mock-treated and cells treated with MIP-1β (compare lane 1 with lane 2). However, we failed to detect any 32P-labeled CCR5Δcyt (Fig. 4 B, lanes 3 and 4). These results suggest that an intact cytoplasmic tail of CCR5 is essential for phosphorylation.
      In summary, our study raises three points: 1) functional CCR5 molecule exists as a multimer; 2) expression of ccr5Δ32 reduces cell surface expression of wild type CCR5; and 3) MIP-1β induces the phosphorylation of CCR5 in its cytoplasmic tail. Taken together, these points suggest that there are complex post-translational regulatory events for CCR5. Relevant to the observation of receptor multimerization, it is clear that CCR5 mutants such as ccr5Δ32 are defective in some specific aspects of post-translational processing that render them aberrantly trapped in the ER (see Fig. 3). However, these defective mutants, nevertheless, maintain an ability to multimerize with wild type CCR5 and through this heterocomplexing retains the wild type moiety into the ER. We believe that this mutant-wild type tethering mechanism explains, in part, why PBMCs from CCR5/ccr5Δ32 individuals have lower levels of cell surface CCR5 when compared with CCR5/CCR5 counterparts (
      • Wu 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.
      ) and whyCCR5/ccr5Δ32 individuals have delayed progression to disease (
      • Dean 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.
      ,
      • Samson 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.
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      • Yi Y.
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      • Collman R.G.
      • Doms R.W.
      • Vassart G.
      • Parmentier M.
      ,
      • Huang Y.X.
      • Paxton W.A.
      • Wolinsky S.M.
      • Neumann A.U.
      • Zhang L.Q.
      • He T.
      • Kang S.
      • Ceradini D.
      • Jin Z.
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      • Kunstman K.
      • Erickson D.
      • Dragon E.
      • Landau N.R.
      • Phair J.
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      • Koup R.A.
      ,
      • Michael N.L.
      • Chang G.
      • Louie L.G.
      • Mascola J.R.
      • Dondero D.
      • Birx D.L.
      • Sheppard H.W.
      ).
      The functional role of phosphorylation on CCR5 is less clear. Likely, this modification governs a step in transducing the MIP-1β signal from the cell surface into the cytoplasm/nucleus. Recently, Amaraet al. (
      • Amara A.
      • Le Gall S.
      • Schwartz O.
      • Salamero J.
      • Montes M.
      • Loetscher P.
      • Baggiolini M.
      • Virelizier J.L.
      • Arenzana-Seisdedos F.
      ) reported that internalization of chemokine receptors (CXCR4 and CCR5), induced by their ligand, contribute to chemokines-mediated inhibition of HIV-1 replication. Interestingly, the cytoplasmic tail of CXCR4 is required for SDF-1-mediated internalization of CXCR4 (
      • Amara A.
      • Le Gall S.
      • Schwartz O.
      • Salamero J.
      • Montes M.
      • Loetscher P.
      • Baggiolini M.
      • Virelizier J.L.
      • Arenzana-Seisdedos F.
      ). Whether phosphorylation of chemokine receptors influences ligand-induced internalization is unknown. Another possibility is that chemokine-stimulated CCR5 phosphorylation can exert entry-unrelated effects that ameliorate HIV-1 infection. Indeed, MIP-1β, MIP-1α, and RANTES have been shown to have unexpected effects on HIV-1 replication in macrophages (
      • Schmidtmayerova H.
      • Sherry B.
      • Burkinsky M.
      ).
      We note that there now exists a plethora of co-receptors for HIV-1 (at least six different ones have been reported; reviewed in Ref.
      • Clapham P.R.
      • Weiss R.A.
      ), suggesting that ligand-based inhibition of HIV-1 infection would be difficult (
      • McKnight A.
      • Wilkinson D.
      • Simmons G.
      • Talbot S.
      • Picard L.
      • Ahuja M.
      • Marsh M.
      • Hoxie J.A.
      • Clapham P.R.
      ). We note additionally that a protective effect to a CCR2 mutation has been recently described (
      • Smith M.W.
      • Dean M.
      • Carrington M.
      • Winkler C.
      • Huttley G.A.
      • Lomb D.A.
      • Goedert J.J.
      • O'Brien T.R.
      • Jacobson L.P.
      • Kaslow R.
      • Buchbinder S.
      • Vittinghoff E.
      • Vlahov R.
      • Buchbinder S.
      • Vittinghoff E.
      • Klahov D.
      • Hoots K.
      • Hilgartner M.W.
      • HGDS.
      • MACS.
      • MHCS.
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      ). Whether the CCR2 mutation shares functional similarities with the CCR5 Δ32 mutation (e.g. producing nonproductive transdominant multimerization or affecting phosphorylation) remains to be explored. However, the natural existence of transdominant negative chemokine receptor mutants might be a general principle that explains differential resistance of certain subpopulations for AIDS and that might help guide future intervention strategies for HIV-1.

      Acknowledgments

      We thank E. Berger for gift of CCR5 plasmid and R. Willey for gift of pCMV/CCR5-Flag construct and for helpful discussions.

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