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A Critical Site in the Core of the CCR5 Chemokine Receptor Required for Binding and Infectivity of Human Immunodeficiency Virus Type 1

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.1905

ISSN

1083-351X

Autores

Salvatore Siciliano, Shawn E. Kuhmann, Youmin Weng, Navid Madani, Martin S. Springer, Janet Lineberger, Renee Danzeisen, Michael D. Miller, Michael P. Kavanaugh, Julie A. DeMartino, David Kabat,

Tópico(s)

Hepatitis C virus research

Resumo

Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (∼98–99% identical) CCR5 of African green monkeys (AGMs) avidly binds β-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections. Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (∼98–99% identical) CCR5 of African green monkeys (AGMs) avidly binds β-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections. human immunodeficiency virus type 1 African green monkey extracellular loop macrophage-tropic simian immunodeficiency virus transmembrane domain glycoprotein fetal bovine serum Dulbecco's modified Eagle's medium macrophage inflammatory protein regulated on activation normal T cell expressed and secreted. 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Virol. 1997; 71: 5003-5011Crossref PubMed Google Scholar, 28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar, 29Bieniasz P.D. Fridell R.A. Aramori I. Ferguson S.S. Caron M.G. Cullen B.R. EMBO J. 1997; 16: 2599-2609Crossref PubMed Scopus (200) Google Scholar, 30Atchison R.E. Gosling J. Monteclaro F.S. Franci C. Digilio L. Charo I.F. Goldsmith M.A. Science. 1996; 274: 1924-1926Crossref PubMed Scopus (279) Google Scholar). Residues of the amino terminus of CCR5 critical to gp120 binding and infection (Y10DINYY15) have since been more precisely defined by other approaches including site-directed mutagenesis (25Ross T.M. Bieniasz P.D. Cullen B.R. J. Virol. 1998; 72: 1918-1924Crossref PubMed Google Scholar,28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar, 32Dragic T. Trkola A. Lin S.W. Nagashima K.A. Kajumo F. Zhao L. Olson W.C. Wu L. Mackay C.R. Allaway G.P. Sakmar T.P. 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Mackay C.R. J. Exp. Med. 1997; 186: 1373-1381Crossref PubMed Scopus (330) Google Scholar) that a monoclonal antibody whose epitope maps to a peptide derived from this domain (2D7) inhibits both infection and 125I-gp120 binding. However, it is not known if 2D7 exerts its inhibitory effects by attaching to a site on CCR5 required for HIV-1 binding or if its binding globally alters CCR5 conformation or sterically interferes with gp120 interaction with another region of the receptor. Therefore, outside of an interaction with the amino terminus of the receptor, other interactions between HIV-1 and CCR5 critical to viral infection are incompletely delineated. Recently, we found a high frequency of heterozygosity for CCR5 substitution polymorphisms in African green monkeys (AGMs), a group of primate species believed to have been infected by immunodeficiency viruses since ancient times (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). These initially identified substitutions predominantly cluster in the amino terminus (D13N and Y14N) and in ECL1 (Q93R and Q93K), and they partially inhibit infections by multiple SIVagmisolates. 2S. E. Kuhmann and D. Kabat, manuscript in preparation. 2S. E. Kuhmann and D. Kabat, manuscript in preparation. Infectivities of R5 HIV-1 isolates were also inhibited by the Y14N and Q93R substitutions in the context of the wild-type AGM CCR5 and by Y14N in the context of human CCR5 (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). However, although wild-type AGM CCR5 is a strong coreceptor for SIV isolates, it is a relatively weak coreceptor for R5 HIV-1 isolates (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). This observation was surprising because rhesus macaque CCR5 is a strong coreceptor for R5 HIV-1 isolates (37Chen Z. Zhou P. Ho D.D. Landau N.R. Marx P.A. J. Virol. 1997; 71: 2705-2714Crossref PubMed Google Scholar) but differs from AGM CCR5 in only three amino acids. Indeed, the only extracellular amino acid substitution that is unique to AGM CCR5 and absent from rhesus and human CCR5s is G163R, which occurs at the juncture of TM4 and ECL2. We now describe evidence that this site is critical for gp120 binding and for infections by all tested R5 isolates of HIV-1. HeLa and HEK293T cells were from the American Type Culture Collection (ATCC, Rockville, MD). HeLa-CD4 (clone HI-J) and HeLa-CD4-CCR5 (clone JC.37) cells were described previously (38Platt E.J. Wehrly K. Kuhmann S.E. Chesebro B. Kabat D. J. Virol. 1998; 72: 2855-2864Crossref PubMed Google Scholar, 39Kabat D. Kozak S.L. Wehrly K. Chesebro B. J. Virol. 1994; 68: 2570-2577Crossref PubMed Google Scholar). HeLa and HeLa-derived cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). HEK293T cells were maintained in the same medium supplemented with glucose (4.5 g/liter). The SF162, JRFL, ADA, and BaL R5 isolates of HIV-1 were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. HIV-1 viruses were passaged in phytohemagglutinin-stimulated human peripheral blood mononuclear cells. Medium was harvested at times of peak reverse transcriptase release, passed through a 0.45-μm pore size filter, aliquoted, and stored at −80 °C. The JRCSF isolate was obtained as an infectious molecular clone, pYK-JRCSF, from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, and transfected into HeLa cells. Culture medium was harvested after 72 h and used to infect HeLa-CD4-CCR5 cells (clone JC.37). Viral supernatants were harvested and filtered as above, and the supernatant from day 3 after infection was used in this study. The rhesus macaque CCR5 expression plasmid was the generous gift of Zhiwei Chen and Preston Marx (Aaron Diamond AIDS Research Center) (37Chen Z. Zhou P. Ho D.D. Landau N.R. Marx P.A. J. Virol. 1997; 71: 2705-2714Crossref PubMed Google Scholar). Constructs containing human and AGM CCR5 were previously described (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). For some experiments, the human and AGM CCR5 plasmids were mutagenized to create the AGM (R163G) and human (G163R) CCR5s by the QuickChange mutagenesis kit (Stratagene, La Jolla, CA) as directed by the manufacturer; for the remaining experiments, mutants with identical coding sequences were created by swapping the BclI to BglII restriction fragment between AGM and human CCR5s. In either case, the entire coding sequence of CCR5 was sequenced to confirm that only the desired mutation was introduced. The chimeric CCR5s were created by splicing AGM and human CCR5 at either the BclI or BglII restriction site as described (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). The human (Y14N) site-directed mutant was described and characterized previously (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). The molecular clone pYU2 was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, and the molecular clone SF162 was from Cecelia Chang-Meyer. The expression and purification of YU2 gp120 are described, with similar procedures used for expression and purification of SF162 gp120. Briefly, the YU2 envelope glycoprotein gp120 was polymerase chain reaction-amplified from proviral DNA using synthetic oligonucleotides designed according to the published sequence. The resulting polymerase chain reaction product was ligated into pSC11 (40Li Y. Hui H. Burgess C.J. Price R.W. Sharp P.M. Hahn B.H. Shaw G.M. J. Virol. 1992; 66: 6587-6600Crossref PubMed Google Scholar), modified to contain a multilinker sequence, to generate pJL23, and was sequence verified. The 3′-antisense primer design appended a FLAG (Eastman Kodak Co.) epitope to the gp120 viral envelope protein following position Arg-498. Plasmid pJL23 was used to generate recombinant vaccinia virus Venv-4 using standard techniques (41Cooper N. Earl P.L. Elroy-Stein O. Moss B. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. John Wiley & Sons, Inc., New York1994: 16.15.1-16.19.9Google Scholar) for large scale expression of soluble gp120-FLAG. 16 T-225 flasks of CV-1 cells were seeded to contain approximately 1–2 × 107 cells/flask on the day of infection. Cells were infected with Venv-4 at a multiplicity of infection of 5 for 2 h in 0.1% bovine serum albumin in phosphate-buffered saline. After infection, cell monolayers were washed twice with phosphate-buffered saline and refed with 50 ml of Opti-MEM (Life Technologies, Inc.) per flask. After approximately 68 h, supernatants were harvested by centrifugation at 6000 rpm for 30 min at 4 °C. Clarified supernatants were supplemented with Triton X-100 (Boehringer Mannheim) to 0.5%, quick-frozen in liquid nitrogen, and stored at −70 °C. Soluble gp120-FLAG proteins were purified by fast protein liquid affinity chromatography using M2-anti-FLAG affinity gel (Kodak) in an HR5/5 column (Amersham Pharmacia Biotech, bed volume ∼1 ml) equilibrated in TBS (50 mm Tris-HCl, pH 8.0, 150 mm NaCl). Culture supernatants (500–1000 ml) were thawed at 37 °C, supplemented to 10 μg/ml each aprotinin and leupeptin (Boehringer Mannheim), filtered through a 0.22-μm filter, and then passed continuously over the M2 column at 1 ml/min at 4 °C for 24–28 h. The resin was washed extensively with TBS, and bound proteins were eluted with 100 μm synthetic FLAG peptide (Kodak) in TBS. Fractions (1 ml) containing gp120-FLAG were identified by Colloidal Blue staining of 10% SDS-polyacrylamide gel electrophoresis gels (Novex, San Diego, CA). Peak fractions were pooled, snap-frozen in liquid nitrogen, and stored at −70 °C. Pooled fractions were separated from synthetic FLAG peptide and TBS by C4 reverse phase chromatography. Samples were loaded onto a 5-cm Vydac C4 analytical column at 1 ml/min in 10% acetonitrile, 0.1% trifluoroacetic acid. Using a 25–50% gradient, gp120 eluted at approximately 36–38% acetonitrile. Samples were collected on dry ice and were immediately lyophilized. Dried samples were resuspended in phosphate-buffered saline. Relative protein concentration determinations were made using a modified gp120 capture enzyme-linked immunosorbent assay (Intracel, Issaquah, WA) using anti-gp120 monoclonal antibody A32 from James Robinson at Tulane. Fractions with peak activity according to enzyme-linked immunosorbent assay were subjected to amino acid analysis for final concentration determinations. BaL gp120 was purified as described previously from the culture medium of Schneider 2 Drosophila cells that were generously donated by Dr. Raymond Sweet (SmithKline Beecham) (42Ivey-Hoyle M. Culp J.S. Chaikin M.A. Hellmig B.D. Matthews T.J. Sweet R.W. Rosenberg M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 512-516Crossref PubMed Scopus (103) Google Scholar). 125I-MIP1α (2200 μCi/mmol) was purchased from NEN Life Science Products. Conditions for competition binding assays using chemokines are similar to those described previously (43Van Riper G. Siciliano S. Fischer P.A. Meurer R. Springer M.S. Rosen H. J. Exp. Med. 1993; 177: 851-856Crossref PubMed Scopus (80) Google Scholar). YU2 gp120 was iodinated by the chloramine-T method according to the procedure of Rollins et al. (44Rollins T.E. Siciliano S. Springer M.S. J. Biol. Chem. 1988; 263: 520-526Abstract Full Text PDF PubMed Google Scholar). Assay conditions for measurement of direct binding of 125I-YU2 gp120 to CCR5-expressing cells, in the presence of 10 nm soluble CD4, and inhibition of binding of 125I-MIP1α to CCR5-expressing cells by YU2 gp120·sCD4 complexes have also been described previously (11Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar). 3S. J. Siciliano, B. L. Daugherty, J. A. DeMartino, and M. S. Springer, manuscript in preparation. For gp120 binding assays done using cells expressing transmembrane-bound CD4, pcDNA3 expression vectors for CD4 and CCR5 were cotransfected into HEK293T cells by the standard DEAE-dextran/chloroquine method (45Chen C.A. Kingston R.E. Okayama H. Selden R.F. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York1994: 9.1.1-9.2.6Google Scholar), except that the cells were plated in flasks that were treated with 0.1 mg/ml poly-l-lysine (Sigma) for 30 min, and no Me2SO shock was used. Cells were seeded 48 h after transfection at 2 × 105cells/well in poly-l-lysine-treated 24-well tissue culture cluster plates. 24 h later cells were incubated with the indicated concentration of BaL gp120 in DMEM, 10% FBS for 30 min at 37 °C.125I-MIP1β (2200 μCi/mmol, NEN Life Science Products) was added to a final concentration of 0.5 nm, and cells were incubated for an additional 30 min. The cells were washed, solubilized in 0.1 n NaOH, and counted in a gamma counter. Background counts were determined on vector-transfected cells and subtracted from the values obtained on CCR5-transfected cells. Counts were then expressed as percent binding by normalizing to values obtained with no added gp120. The assay to determine infectivities by R5 HIV-1 isolates was performed as described previously (28Kuhmann S.E. Platt E.J. Kozak S.L. Kabat D. J. Virol. 1997; 71: 8642-8656Crossref PubMed Google Scholar). Briefly, coreceptors were transiently expressed in HeLa-CD4 (clone HI-J) cells by the calcium phosphate transfection method (45Chen C.A. Kingston R.E. Okayama H. Selden R.F. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York1994: 9.1.1-9.2.6Google Scholar). 48 h post-transfection the cultures were trypsinized and plated at 1.5 × 104 cells/well of a 24-well cluster plate for HIV-1 infection. 72 h post-transfection cells were pretreated with DEAE-dextran (8 μg/ml) at 37 °C for 20 min and then incubated with 0.2 ml of virus diluted in DMEM, 0.1% FBS at 37 °C. After 2 h the cells were fed with 1 ml of DMEM, 10% FBS, and incubated at 37 °C for 3 days. The cells were then fixed in ethanol, and infected foci were visualized by an immunoperoxidase assay (46Chesebro B. Wehrly K. J. Virol. 1988; 62: 3779-3788Crossref PubMed Google Scholar), using as primary antibody the 0.45-μm filtered supernatant from the anti-p24 hybridoma 183-H12–5C (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health). Stained foci were counted with a dissecting microscope under diffuse illumination, and values were normalized to those obtained using the same virus stock in the same experiment on cells transfected with wild-type human CCR5. Xenopus laevis oocytes were collected and prepared as described previously (47Madani N. Kozak S.L. Kavanaugh M.P. Kabat D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8005-8010Crossref PubMed Scopus (45) Google Scholar). CCR5 cDNAs were subcloned into the oocyte expression vector pOG-1 at a site between 5′- and 3′-untranslated Xenopus β-globin sequences. Kir 3.1 and CCR5 cRNAs were prepared as described previously, and oocytes were microinjected with 5–50 ng of capped cRNA (47Madani N. Kozak S.L. Kavanaugh M.P. Kabat D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8005-8010Crossref PubMed Scopus (45) Google Scholar). Electrophysiological recording was done by two-electrode voltage clamp 2–5 days after cRNA injection as described (47Madani N. Kozak S.L. Kavanaugh M.P. Kabat D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8005-8010Crossref PubMed Scopus (45) Google Scholar). Briefly, the oocytes were clamped in a small chamber continually perfused with high K+ Ringer's solution (100 mm KCl, 2 mm NaCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.5), and recombinant human chemokines (Peprotech, Rocky Hill, NJ) were applied by bath perfusion. The holding potential was set at −30 mV, and current-voltage records were obtained during 250-ms voltage jumps to potentials between +40 and −100 mV. Desensitization kinetics were determined by least squares fit to single exponential functions. Binding of the mouse monoclonal antibody 2D7 was determined on HEK293T cells transfected as for gp120 binding (above). 72 h post-transfection cells were incubated with 2.5 μg/ml 2D7 (PharMingen, San Diego, CA) in DMEM, 10% FBS for 45 min at 37 °C, followed by goat anti-mouse IgG serum (Organon Teknika Corp., Durham, NC) at a 1:400 dilution for 45 min, followed by125I-protein A (0.4 μCi/ml, 2 to 10 μCi/μg; NEN Life Science Products) for 45 min. The cells were then washed, solubilized in 0.1 n NaOH, and counted in a gamma counter. Background counts were determined on vector-transfected cells and subtracted from the values obt

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