A PIJfATIVE chemokine receptor that we previously cloned and termed LESTR 1 has recently been shown to function as a coreceptor (termed fusin) for lymphocyte-tropic HIV-1 strains 2 • Cells expressing CD4 became permissive to infection with T -cellline-adapted HIV-1 strains of the syncytium-i.nducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the identification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the naturaJ ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new cbemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 eDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1a and MIP-1~, which were shown previously to prevent infection with primary, monocyte-tropic viruses 3 , are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS 4• LESTR (leukocyte-expressed seven-transmembrane-domain receptor) is an orphan receptor with structural similarity to chemokine receptors. Despite extensive testing of a large number of chemokines, the ligand for LESTR remained elusive 1 • Murine SDF-1 was described as a factor that is produced by bonemarrow stromal cells and shown to induce proliferation of B-cell progenitorsM as well as recruitment of T cells 7 • The human homologue, which was cloned subsequently, is virtually identical to murine SDF-1 (see Methods). SDF-1 is a CXCchemokine with the typical four-cysteine motif and the first two cysteines separated by one amino acid 8 • When human SDF-1 was tested on the CH0-1C2 clone which stably expresses LESTR, a transient rise of cytosolic free Ca 2 + ([Ca 2 +];) was observed (Fig. 1a). This response, which is characteristic of the action of chemokines on blood leukocytes, was not observed with parental CHO cells. Other chemokines, including RANTES (for regulation-upon-activation, normal T expressed and secreted) macrophage inflammatory protein (MIP), MIP-1o: and MIP-1~, were not active. Monocytes, neutrophils and phytohaemagglutinin (PHA)-activated peripheral-blood lymphocytes (PBLs) were also stimulated by SDF-1, as shown by [Ca 2 +]; changes and chemotaxis (Fig. 1b, d). Real-time recordings of Ca 2 + mobilization after sequential stimulation are a reliable way to assess receptor usage by chemokines 8 • Stimulation with a chemokine (at saturating concentrations) causes receptor desensitization, and no response is observed when the cells are restimulated within a short time by a chemokine acting on the same receptor. As shown in Fig. lc, monocytes stimulated with SDF-1 remained fully responsive to subsequent stimulation with ...

A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.

Ligation of CCR5 by the CC chemokines RANTES, MIP-1α or MIP-1β, and of CXCR4 by the CXC chemokine SDF-1α, profoundly inhibits the replication of HIV strains that use these coreceptors for entry into CD4+ T lymphocytes. The mechanism of entry inhibition is not known. We found a rapid and extensive downregulation of CXCR4 by SDF-1α and of CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via Gi-proteins. Removal of SDF-1α led to rapid, but incomplete surface reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1α–induced receptor downregulation and decreased the potency of SDF-1α as inhibitor of HIV replication. Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.