CXCR4 Sequences Involved in Coreceptor Determination of Human Immunodeficiency Virus Type-1 Tropism
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.15007
ISSN1083-351X
AutoresZixuan Wang, Joanne F. Berson, Tianyuan Zhang, Yin-Hua Cen, Yi Sun, Matthew Sharron, Zhao-hai Lu, Stephen C. Peiper,
Tópico(s)T-cell and B-cell Immunology
ResumoThe interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism. The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. 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These findings provide further insight into the complexity of the interaction between gp120 and coreceptors during the progression of infection and furnish a model coreceptor system that could be widely applicable to the study of HIV-1 entry into target cells and to virally mediated gene therapy of AIDS. Constructs encoding wild type CXCR4 and CCR5 in the pcDNA3 vector described previously were used as templates for site-directed mutagenesis using the Chameleon double-stranded site-directed mutagenesis kit (Stratagene, San Diego, CA). Clones containing the programmed mutation(s) were identified by nucleotide sequence analysis of the targeted region, and the nucleotide sequence of the entire open reading frame was confirmed prior to analysis in fusion assays to exclude the possible introduction of extraneous mutations. A chimeric receptor containing the N-terminal extracellular domain of CCR5 and the complementary region of CXCR4, designated 5444, and a battery of chimeras composed of CXCR4 and CXCR2, both of which were prepared by polymerase chain reaction-ligation-polymerase chain reaction as described previously, were also used as templates for site-directed mutagenesis (34Lu Z.-H. Berson J. Cen Y.-H. Turner J.D. Zhang T.-Y. Sharron M. Jenks M.H. Wang Z.-X. Kin J. Rucker J. Hoxie J.A. Peiper S.C. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6426-6431Crossref PubMed Scopus (168) Google Scholar). The coreceptor function of wild type chemokine receptors, variants containing point mutations, and chimeras was determined using a modified fusion assay (36Nussbaum O. Broder C.C. Berger E.A. J. Virol. 1994; 68: 5411-5422Crossref PubMed Google Scholar) employing a luciferase reporter gene as described previously (33Doranz B.J. Lu Z.-H. Rucker J. Zhang T.-Y. Sharron M. Cen Y.-H. Wang Z.-X. Guo H.-H. 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Fusion was determined by measuring luciferase activity 5–6 h postmixing. Viral infection assays were performed as described previously using recombinant viruses containing clonedenv genes and a luciferase reporter gene (48Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar, 49Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1111) Google Scholar). Viral stocks were prepared as described previously by infecting 293T cells with plasmids encoding the IIIB, ADA, or BaL Env proteins and the NL4–3 luciferase virus backbone (48Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar, 49Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1111) Google Scholar). U87-MG target cells were seeded in 24-well plates and transfected with plasmids encoding CD4 and wild type or mutant coreceptors. Following incubation for 24 h to permit transient expression, the cells were infected with viral stocks in the presence of 4 μg of polybrene/ml in a total volume of 500 μl. Three days postinfection, an additional 0.5 ml of medium was added. Four days postinfection, the cells were harvested by resuspension in 150 μl of 0.5% Triton X-100 in phosphate-buffered saline, and 50–75-μl aliquots were assayed for luciferase activity using commercial reagents (Promega, Madison, WI) in a Wallac 1450 Microbeta luminometer. The expression of chemokine receptor chimeras and point mutants on the surface of transfectants was measured by flow cytometry using monoclonal antibodies to CXCR4 (12G5), CCR5 (12D1 and R&D 45529; R&D Systems, Minneapolis, MN), and CXCR2 (10H2). Cells were stained at room temperature with the appropriate monoclonal antibody, washed, incubated with a secondary antibody labeled with phycoerythrin, and analyzed using an Elite flow cytometer (Coulter Electronics, Inc., Miami, FL). Previous studies using CXCR4/CXCR2 chimeras have demonstrated the importance of ECL2 of CXCR4 in coreceptor activity with Env glycoproteins encoded by IIIB (T-tropic) and 89.6 (dual tropic) (34Lu Z.-H. Berson J. Cen Y.-H. Turner J.D. Zhang T.-Y. Sharron M. Jenks M.H. Wang Z.-X. Kin J. Rucker J. Hoxie J.A. Peiper S.C. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6426-6431Crossref PubMed Scopus (168) Google Scholar). In order to dissect the motifs that are involved in this function, an aggressive mutagenesis strategy was initially focused on charged amino acid residues in ECL2 and adjacent transmembrane-spanning helices. Alanine (Ala) scanning mutagenesis was performed to identify charged residues in this domain of CXCR4 that may be involved in the interaction with dual tropic and T-tropic envelope glycoproteins. This loop contains 5 acidic and 2 basic amino acid residues, and the fourth transmembrane-spanning domain (tm4) contains 1 acidic residue, as depicted in Fig.1. Variants were prepared in which the charged residues were individually converted to Ala by site-directed mutagenesis. Fusion coreceptor activity of the Ala-scanning mutants with IIIB and 89.6 Env glycoproteins did not differ significantly from that of the wild type receptor (data not shown), indicating that no single residue was critical to coreceptor activity. There was a suggestion that removal Glu-179 and Asp-182 may result in a subtle but consistent enhancement of coreceptor activity. Since 6 of 13 residues in the proximal region of ECL2 of CXCR4 are charged, it was reasoned that several may contribute to a structure that interacts with Env. To test this possibility, variants in which multiple charged residues were converted to neutral ones were prepared and analyzed for coreceptor function (data not shown). Glu-179 was replaced with Gln in order to maintain potential hydrophilic boundaries of tm4 while removing the net charge. CXCR4-E179Q/D181A/D182N showed a moderate decrease in utilization by IIIB and 89.6. The conversion of Asp-187 and Lys-188 to Ala, with or without D193A, did not have a dramatic effect on coreceptor activity. Surprisingly, a variant in which all acidic and one of the basic (Arg-189) residues were converted to neutral amino acids (E179Q/D181A/D182N/R188A/D193A) retained significant coreceptor activity with IIIB (∼50%) and 89.6 (∼25%). The absence of a significant impact of remodeling the conformation of ECL2 by the removal of 6 of 7 charged amino acids on the fusion cofactor activity for dual tropic and T-tropic Env suggests that they are not directly involved in the structure that associates with Env. To test the possibility that noncharged residues participate in this structure, Phe-189 and Tyr-190 were replaced with Ala, and a hydrophobic stretch in the distal portion of ECL2 was interrupted by converting Val-197 to Asn. Neither of these mutations significantly altered fusion coreceptor function with IIIB and 89.6 (data not shown). The excess of acidic residues in ECL2 of CXCR4 is contrasted by an excess of basic amino acids in the corresponding domain of CCR5, which does not support fusion with T-tropic Env (33Doranz B.J. Lu Z.-H. Rucker J. Zhang T.-Y. Sharron M. Cen Y.-H. Wang Z.-X. Guo H.-H. Du J.-G. Accavitti M.A. Doms R.W. Peiper S.C. J. Virol. 1997; 71: 6305-6314Crossref PubMed Google Scholar). To determine whether the net charge of ECL2 is critical to determining the coreceptor activity of CXCR4 to include T-tropic and dual tropic but not M-tropic Env, each acidic residue in this domain was converted individually to Lys. Analysis of these charge conversion mutants failed to reveal a significant alteration in coreceptor activity with IIIB and 89.6 (data not shown). Surprisingly, one of these variants, CXCR4-D193K, seemed to have minimal enhancement of utilization by 89.6. Conversion of Asp-171, which is located in tm4, to Lys resulted in a marked loss of coreceptor function with the 89.6 (6% of wild type CXCR4) and IIIB (16%) Env glycoproteins. However, this variant was utilized as a fusion coreceptor by the BK132 and DH12 Env glycoproteins at levels approximately 25% of wild type CXCR4 (data not shown). A variant in which multiple acidic amino acid residues were switched to Lys, CXCR4-E179K/D181K/D182K, was produced to mimic the charge of the proximal region in ECL2 of CCR5. This mutant demonstrated a dramatic loss of function. Neither of these variants were detected on the cell surface by immunofluorescent staining (data not shown), suggesting that the mutations interfered with intracellular trafficking. The difference in charge between ECL2 of CCR5 and CXCR4 and the frequent acquisition of positively charged residues in the V3 loop of Env upon conversion from M- to T-tropism (43Fouchier R.A.M. Groenink M. Kootstra N.A. Tersmette M. Huisman H.G. Miedema F. Schuitemaker H. J. Virol. 1992; 66: 3183-3187Crossref PubMed Google Scholar, 44De Jong J.J. De Ronde A. Keulen W. Tersmette M. Goudsmit J. J. Virol. 1992; 66: 6777-6780Crossref PubMed Google Scholar, 45Jansson M. Popovic M. Karlsson A. Cocchi F. Rossi P. Albert J. Wigzell H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15382-15387Crossref PubMed Scopus (103) Google Scholar) raised the possibility that a negatively charged ECL2 is required to limit the coreceptor function of CXCR4 to T-tropic Env and that changing the charge of this domain could be associated with a gain of coreceptor utilization by M-tropic Env. To test this possibility, the Ala scanning and charge conversion point mutants described above were tested in cell-cell fusion assays with JRFL. None of the CXCR4 variants containing charge conversion mutations were found to acquire coreceptor activity with this M-tropic Env (Fig.2 A ). Parallel analysis of the Ala-scanning mutants, also shown in Fig. 2 A , revealed that a single point mutant, CXCR4-D187A, functioned as a coreceptor for JRFL, demonstrating approximately 25% of the activity of CCR5. This point mutant was also found to be utilized as a fusion coreceptor by other M-tropic Env, including ADA, Bal, and SF162 (data not shown). Analysis of CXCR4 variants with multiple mutations for M-tropic coreceptor activity revealed that variants containing the D187A mutation exhibited coreceptor activity with JRFL (data not shown). This activity was not altered significantly by the addition of R188A, D193A, F189A/Y190A, or V197N, but it was diminished by E179Q/D181A/D182A, E179Q/D181A/D182A/R188A, and E179Q/D181A/D182A/R188A/D193A. The acquisition of M-tropic coreceptor activity was not observed in variants lacking D187A. To determine the requirements at amino acid residue 187 of CXCR4 for the maximum acquisition of M-tropic coreceptor activity, saturation mutagenesis was performed. As shown in Fig. 2 B , the conversion of CXCR4-Asp-187 to Val, Phe, and Ser was associated with the acquisition of coreceptor activity with JRFL, whereas replacement with Asn resulted in limited M-tropic coreceptor activity, which was absent when Lys was substituted. CXCR4-D187V consistently demonstrated a significant level of fusion coreceptor activity with M-tropic Env, with a mean value that was greater than 50% of wild type CCR5. However, these mutations had minimal effects on the coreceptor activity of CXCR4 with IIIB and 89.6 (data not shown). The M-tropic coreceptor activity of CXCR4-D187V could be inhibited by the addition of SDF-1, the ligand for CXCR4, and ALX40–4C, a pharmacologic inhibitor of CXCR4, to the fusion reaction, but not by AO
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