Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages

Abstract

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.

FIG. 1.

Effects of HCC-1[9-74] and RANTES on YU2 replication. Prestimulated human PBMCs were infected with HIV-1 YU2 virus stocks containing 20 ng of p24 antigen in the presence of the indicated concentrations of HCC-1[9-74] (A) or RANTES (B). Supernatant was harvested at regular intervals and analyzed for RT activity, as described in Materials and Methods. The results shown were obtained with PBMCs from one donor in a single experiment. The study was repeated eight times with different batches of chemokine. Usually, HCC-1[9-74] and RANTES blocked YU2 replication with comparable efficiencies (data not shown). PSL, photon-stimulated light emission; ×, uninfected cells.

FIG. 2.

Effects of HCC-1[9-74] on replication of different HIV-1 isolates in human PBMCs. Prestimulated PBMCs were exposed to the various HIV-1 isolates in the presence of the indicated concentrations of the chemokine. Virus stocks containing 20 ng of p24 antigen were used for infection. The results obtained with PBMCs from one donor in a single experiment are shown. Similar results were obtained in two independent experiments with PBMCs from different donors. Cells were cultured and virus production was measured by RT assay, as described in the legend to Fig. 1. PSL, photon-stimulated light emission.

FIG. 3.

Low concentrations of HCC-1[9-74] can increase viral replication. PBMCs from a single donor were infected in triplicate with normalized amounts (20 ng) of the R5-tropic YU2 isolate in the presence of the indicated amounts of HCC-1[9-74]. The data represent the average values and standard deviations. Cells were cultured and virus production was measured by RT assay, as described in the legend to Fig. 1. Similar results were obtained in independent experiments and with the R5-tropic JR-CSF isolate. PSL, photon-stimulated light emission.

FIG. 4.

The effects of low doses of HCC-1[9-74] on YU2 replication depend on the PBMC donor. PBMCs from donors A and B were infected, and virus production was measured by the RT assay, as described in the legend to Fig. 1. Average values obtained from triplicate infections are shown. Donor-dependent variations were confirmed in five independent experiments with PBMCs from 10 additional donors and different virus stocks. PSL, photon-stimulated light emission.

FIG. 5.

HCC-1[9-74] inhibits HIV-1 replication in macrophages. Macrophages were isolated from fresh blood by using lymphocyte separation medium as described in the Materials and Methods section. Cells were infected with virus stocks containing 1 ng of p24 antigen, and RT activities were measured as described in the legend to Fig. 1. Average values obtained from triplicate infections are shown. Comparable results were obtained in two independent experiments with MDMs derived from different donors. PSL, photon-stimulated light emission.

FIG. 6.

Effects of HCC-1 length variants on HIV-1 YU2 replication in PBMCs. (A) N-terminal sequences of the HCC-1 variants analyzed. (B) Human PBMCs were infected in triplicate with YU2 in the presence of the indicated chemokine concentrations (an HCC-1[9-74] concentration of 1 μg/ml corresponds to 128 nM). The data represent the average peak RT activity measured in cell culture supernatants 22 days after infection. The RT activity detected in the absence of chemokine was considered 100% replication efficiency. (C) P4-CCR5 cells were infected with the YU2 strain in the presence of the various chemokines, and infectivity was assayed as described previously (10). Average values obtained from triplicate infections are shown. ×, RANTES.

FIG. 7.

HCC-1[9-74] binds to ECL-2 of R5. (A) 293T cells were transfected with expression plasmids encoding R5 and CCR2b chimeras. Chimeras are designated by their extracellular domains as described previously (20): 5555, wild-type R5; 5525, a chimera containing the ECL-2 region of CCR2b; 2252, a CCR2b chimera containing the ECL-2 domain of R5. At 48 h posttransfection, the cells were harvested and processed for the binding assay, as described in Materials and Methods. The results of the experiment shown are representative of two performed in triplicate. The data are represented as means ± standard errors of the means. (B) Binding of HCC-1[9-74] to 293 cells stably transfected with R5 preincubated with MAbs that interact with the N terminus (MAb CTC5) or the ECL-2 region (MAbs 2DZ and 45531) of R5 (20, 45). The results of the experiment shown are representative of two performed. mIg, membrane immunoglobulin.

FIG. 8.

Down-regulation of R5 from the surfaces of stable transfected GHOST (03) Hi-5 cells. HOS cells expressing CD4 and high levels of R5 were incubated with the indicated concentrations of the different chemokines. Cell surface R5 expression was detected with R5-specific MAb 182 and analyzed by FACS analysis. Values give the percentages of mean fluorescence intensity.

FIG. 9.

Effects of chemokines on the subcellular localization of R5-GFP. HeLa cells transiently expressing R5-GFP were treated with medium alone (A) or with medium containing 100 μg of either HCC-1[9-74] (B), RANTES (C), or HCC-1 (D) per ml for 45 min at 37°C. After fixation the cells were examined by fluorescence microscopy.