Determinants of HIV-1 Coreceptor Function on CC Chemokine Receptor 3
1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês
10.1074/jbc.272.33.20420
ISSN1083-351X
AutoresGhalib Alkhatib, Edward A. Berger, Philip M. Murphy, James E. Pease,
Tópico(s)T-cell and B-cell Immunology
ResumoThe chemokine receptors CXCR4, CCR2b, CCR3, and CCR5 are cell entry coreceptors for HIV-1. Using an HIV-1 envelope (Env)-dependent cell-cell fusion model of entry, we show that CCR3 can interact with Envs from certain macrophage (M)-tropic strains (which also use CCR5), T cell line (TCL)-tropic laboratory-adapted strains (which also use CXCR4), and a dual-tropic primary isolate (which also uses CCR2b, CCR5, and CXCR4). Paradoxically, CCR1 is the closest homologue to CCR3 (63% amino acid identity), but lacked HIV-1 coreceptor activity. These results confirm and extend previous reports. Replacing the N-terminal segment of CCR3 with that of CCR1 abolished activity of the resulting chimera for M-tropic and TCL-tropic Envs, but not for the dual-tropic Env. Replacing extracellular loop 2 of CCR3 with that of CCR1 abolished activity for TCL-tropic Envs, but not for M- and dual-tropic Envs. A chimera containing all four extracellular regions of CCR3 on a backbone of CCR1 lacked any activity. Env-CCR3 interactions were strongly inhibited by the major CCR3 ligand eotaxin, but weakly or not at all by other CCR3 ligands. With primary macrophages, eotaxin induced transient calcium flux and partially inhibited fusion with cells expressing M-tropic Envs. We conclude that specificity determinants for different Envs are located in shared and distinct extracellular regions of CCR3, the transmembrane/cytoplasmic domains make major contributions to coreceptor function, and CCR3 may be used by certain HIV-1 strains as a cell fusion factor on macrophages. The chemokine receptors CXCR4, CCR2b, CCR3, and CCR5 are cell entry coreceptors for HIV-1. Using an HIV-1 envelope (Env)-dependent cell-cell fusion model of entry, we show that CCR3 can interact with Envs from certain macrophage (M)-tropic strains (which also use CCR5), T cell line (TCL)-tropic laboratory-adapted strains (which also use CXCR4), and a dual-tropic primary isolate (which also uses CCR2b, CCR5, and CXCR4). Paradoxically, CCR1 is the closest homologue to CCR3 (63% amino acid identity), but lacked HIV-1 coreceptor activity. These results confirm and extend previous reports. Replacing the N-terminal segment of CCR3 with that of CCR1 abolished activity of the resulting chimera for M-tropic and TCL-tropic Envs, but not for the dual-tropic Env. Replacing extracellular loop 2 of CCR3 with that of CCR1 abolished activity for TCL-tropic Envs, but not for M- and dual-tropic Envs. A chimera containing all four extracellular regions of CCR3 on a backbone of CCR1 lacked any activity. Env-CCR3 interactions were strongly inhibited by the major CCR3 ligand eotaxin, but weakly or not at all by other CCR3 ligands. With primary macrophages, eotaxin induced transient calcium flux and partially inhibited fusion with cells expressing M-tropic Envs. We conclude that specificity determinants for different Envs are located in shared and distinct extracellular regions of CCR3, the transmembrane/cytoplasmic domains make major contributions to coreceptor function, and CCR3 may be used by certain HIV-1 strains as a cell fusion factor on macrophages. 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Human HeLa cells and murine NIH 3T3 cells were obtained from the American Type Culture Collection (Rockville, MD). For preparation of primary macrophages (38Lazdins J.K. Woods-Cook K. Walker M. Alteri E. AIDS Res. Hum. Retroviruses. 1990; 6: 1157-1161Crossref PubMed Scopus (38) Google Scholar), elutriated monocytes were obtained from healthy blood donors under an approved protocol by the Dept. of Transfusion Medicine, Clinical Center, National Institutes of Health. Monocytes were washed and resuspended in Dulbecco's modified Eagle's medium plus 10% human AB serum (Advanced Biotechnologies, Columbia, MD) and then incubated on bacteriological plates for 2 weeks, with fresh medium added every 7 days. The cells were recovered by incubation at 4 °C for 15 min, then cryopreserved in 90% fetal bovine serum, 10% Me2SO in liquid nitrogen until use. Macrophages (107/ml) were loaded with Fura-2 (Molecular Probes, Eugene, OR) as described previously (27Combadiere C. Ahuja S.K. Murphy P.M. J. 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Switch sites for the chimeras were chosen so as to independently exchange each of the four extracellular domains of CCR1 and -3. Correct sequence and orientation in the vaccinia expression plasmid pSC59 were verified for all constructs. Fusion between Env-expressing effector cells and CD4/coreceptor-expressing target cells was monitored by a vaccinia-based reporter gene assay for cell-cell fusion (40Nussbaum O. Broder C.C. Berger E.A. J. Virol. 1994; 68: 5411-5422Crossref PubMed Google Scholar). Recombinant plasmids containing coreceptor constructs downstream of the vaccinia synthetic promoter were transfected into target NIH 3T3 cells by lipofection using DOTAP (Boehringer Mannheim). The vaccinia promoter was activated by coinfection of the transfected cells with recombinant vaccinia viruses vCB-3 encoding human CD4 (41Broder C.C. Dimitrov D.S. Blumenthal R. Berger E.A. Virology. 1993; 193: 483-491Crossref PubMed Scopus (101) Google Scholar) and vTF7-3 encoding T7 RNA polymerase (42Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1875) Google Scholar). Surface expression of the flag epitope was verified by FACS using the M2 monoclonal antibody according to the recommendations of the manufacturer (Eastman Kodak Co., Rochester, NY). Effector HeLa cells were co-infected with vCB-21R containing theEscherichia coli lacZ gene under control of the T7 promoter (43Alkhatib G. Broder C.C. Berger E.A. J. Virol. 1996; 70: 5487-5494Crossref PubMed Google Scholar) plus one of the following Env-encoding vaccinia viruses (44Broder C.C. Berger E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9004-9008Crossref PubMed Scopus (134) Google Scholar): vCB-41, LAV; vCB-36, RF; vCB-43, Ba-L; vCB-28, JR-FL; vCB-32, SF-162; vCB-39, ADA; vCB-16, a nonfusogenic uncleavable (Unc) Env. The IIIB (BH8) Env was expressed using vSC60.2 The 89.6 Env (cloned into pSC59) was expressed by lipofection of HeLa cells and infection with vCB-21R. Where indicated, infections of HeLa cells were performed in the presence of 40 μg/ml cytosine arabinoside (araC). Effector HeLa cells and target NIH 3T3 cells were incubated separately overnight at 31 °C to allow synthesis of vaccinia virus-encoded proteins, then washed, and resuspended at 106 per ml. Mixtures (duplicate) were prepared containing equal numbers (1 × 105) of effector and target cells and incubated for 2.5 h at 37 °C in the presence or absence of recombinant chemokines. Reactions were terminated by addition of 0.5% Nonidet P-40. Fusion was scored by colorimetric assay of lysates for β-galactosidase activity. When primary macrophages were used as targets, macrophages were infected with vTF7-3, and fusion with Env-expressing HeLa effector cells was analyzed as described above. CCR3 exhibited strong fusion activity with all M-tropic Envs tested (Ba-L, JR-FL, SF-162, and ADA), two of three laboratory-adapted TCL-tropic Envs tested (the closely related IIIB and LAV, but not RF), and the dual-tropic primary isolate 89.6 Env (Fig. 1). In marked contrast, CCR1 was inactive with all Envs tested despite similar levels of surface expression, as determined by FACS with the M2 antibody recognizing the epitope tag (see Fig. 1 legend).Figure 1HIV-1 Env specificity of CCR1 and CCR3.Cell fusion was monitored between target NIH 3T3 cells coexpressing CD4 and either epitope-tagged CCR3, epitope-tagged CCR1, or neither coreceptor (None), as specified in the upper leftof each part of panel A or on the x axis ofpanel B, with effector HeLa cells expressing Envs from the HIV-1 strains indicated on the x axis of panel Aor from the dual-tropic strain 89.6 in panel B. Data for the 89.6 Env are plotted separately because the vector used differed from all the other Envs tested (plasmid versus recombinant vaccinia viruses). The data are the mean ± S.E. from one experiment representative of greater than 5 separate experiments, each carried out in duplicate. In a separate experiment to measure surface expression of coreceptors by FACS, the relative mean fluorescence values were 127 for CCR1 and 116 for CCR3 using the monoclonal antibody M2 directed against the FLAG epitope and 11 using a negative control isotype-matched monoclonal antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To map Env specificity determinants on CCR3, we analyzed the HIV-1 coreceptor activity of eight CCR1/CCR3 chimeras for the LAV, IIIB, 89.6, Ba-L, JR-FL, and ADA Envs (Figs. 2 and3). Since the extracellular regions of CCR1 and CCR3 contain the majority of amino acid differences between the two molecules (Fig. 2) and are the most likely regions to directly contact Env, we switched only these regions, singly and in combinations. The boundaries for each exchange were based on hydropathy analysis of the primary sequences, and were limited precisely to the predicted extracellular hydrophilic domains by appropriate design of polymerase chain reaction amplimers (Figs. 2 and 3). All chimeras were expressed on the cell surface at levels similar to those for CCR1 and CCR3 as determined with the M2 anti-FLAG monoclonal antibody (see Fig.3 legend).Figure 3HIV-1 coreceptor activity and specificity of CCR1/CCR3 chimeric receptors. Each graph shows the fusion activities of target NIH 3T3 cells expressing CD4 plus an epitope-tagged chimeric receptor when mixed with effector HeLa cells expressing the Envs indicated on the x axis at thebottom. Schemas of the sequence composition of each construct tested are given to the right of the corresponding graph (white, CCR3 sequence specificity; black, CCR1 sequence specificity). Each construct also contained the FLAG epitope tag at the amino terminus, inserted between the first two amino acids. For each Env, fusion data are expressed as the values obtained with the indicated chimera compared with wild type CCR3; the low value obtained with the Unc Env was subtracted in calculating the data shown. The results represent mean values of duplicate samples (error bars denote standard error of the population); the results are representative of at least three separate experiments with each construct. Comparable surface expression of each construct was verified by FACS analysis with the monoclonal antibody M2 directed against the FLAG epitope. In one experiment, the relative mean fluorescence values using the M2 antibody were: CCR1, 127; CCR3, 116;CHI 1, 196; CHI 2, 165; CHI 3, 153; and CHI 4, 183; the value using a control isotype-matched monoclonal antibody was 11. In another experiment, the values with the M2 antibody were: CCR1, 73; CCR3, 63; CHI 4, 33; CHI 5, 38; CHI 6, 42; CHI 7, 40; and CHI 8, 135; background staining for the isotype control antibody, 7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the first set of chimeras, we replaced extracellular domains of CCR3 with the corresponding domains of CCR1 one at a time (chimeras CHI 1 through CHI 4). We observed that the chimera containing the N-terminal segment of CCR1 (chimera CHI 1) had <20% of the fusion activity of wild type CCR3 for Envs from the laboratory-adapted TCL-tropic strains (IIIB and LAV), as well as for Envs from the M-tropic strains (JR-FL, ADA, and Ba-L). This suggests that the N-terminal segment of CCR3 is necessary for interaction with the laboratory-adapted TCL-tropic and the M-tropic Envs. However, it is not sufficient since chimera CHI 4, which contains only the third extracellular loop of CCR1, also exhibited a marked reduction in fusion activity for the same Envs. Compared with the M- and TCL-tropic Envs tested, Env from the dual-tropic primary isolate 89.6 was exceptional in exhibiting a high level (80%) of the fusion activity of wild type CCR3 when tested with chimera CHI 1, although, like the other Envs, it also interacted poorly with chimera CHI 4 (20% of wild type CCR3). Thus, different HIV-1 strains can finely discriminate not only among different chemokine receptors, but also among different domains of the same receptor. This point is further supported by the phenotype of chimera CHI 3, which contains the second extracellular loop of CCR1. While this chimera exhibited fusion activity at levels comparabl
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