Research ArticleCELL BIOLOGY

Targeting CCR5 trafficking to inhibit HIV-1 infection

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Science Advances  16 Oct 2019:
Vol. 5, no. 10, eaax0821
DOI: 10.1126/sciadv.aax0821
  • Fig. 1 Differential anterograde transport of CCR5 and TNF.

    (A) Synchronized transport of CCR5 (top) and TNF (bottom) in HeLa cells stably expressing Str-KDEL_SBP-EGFP-CCR5 or Str-KDEL_TNF-SBP-EGFP. Trafficking was induced by addition of biotin at 0 min. Scale bar, 10 μm. (B) Dual-color imaging of the synchronized transport of SBP-EGFP-CCR5 and TNF-SBP-mCherry transiently coexpressed in HeLa cells. Streptavidin-KDEL was used as an ER hook. Release from the ER was induced by addition of biotin at 0 min. Scale bar, 10 μm. (C) Magnification (×2.8) of the Golgi complex region is displayed. Scale bar, 10 μm. (D) Kinetics of arrival of CCR5 (magenta) or TNF (cyan) to the cell surface after release from the ER measured by flow cytometry. Ratio of cell surface signal divided by GFP intensity was used for normalization. a.u., arbitrary units. The mean ± SEM of three experiments is shown. See also movies S1 to S3.

  • Fig. 2 Identification of molecules inhibiting CCR5 transport by high-content differential screening.

    (A) Outline of the chemical screening strategy. (B) Micrographs from screening plates showing controls. DMSO without biotin and DMSO with biotin correspond to the conditions where no transport and normal transport occurred, respectively. HeLa cells stably expressing Str-KDEL_SBP-EGFP-CCR5 and Str-KDEL_TNF-SBP-EGFP are displayed in the top and bottom panels, respectively. Cargo at the plasma membrane was detected using anti-GFP antibodies on nonpermeabilized cells. (C and D) Clustering of molecules obtained after bioinformatics analysis of the Prestwick (C) and NCI (D) chemical library screening of CCR5 secretion. Below the dendrogram, each bar corresponds to a well of the plate. Molecules identified as hits were shifted one lane below for better visualization. BFA, brefeldin A; Noco, nocodazole. (E) Micrographs showing the three classes of hits detected. CCR5 and TNF hit corresponds to a molecule affecting transport of both CCR5 and TNF. CCR5-specific hit or TNF-specific hit corresponds to a molecule inhibiting only CCR5 or only TNF transport. HeLa cells stably expressing Str-KDEL_SBP-EGFP-CCR5 and Str-KDEL_TNF-SBP-EGFP are displayed in the top and bottom panels, respectively. Cargo at the plasma membrane was detected using anti-GFP antibodies on nonpermeabilized cells.

  • Fig. 3 Three small molecules inhibit the transport of CCR5 and do not affect transport of CCR1 or CXCR4.

    (A) Kinetics of synchronized transport of three chemokine receptors—CCR5 (black), CCR1 (red), and CXCR4 (green)—using the RUSH assay. Trafficking was induced by addition of biotin at time 0. Surface expression of CCR5, CCR1, and CXCR4 was measured by flow cytometry using an anti-GFP antibody. Ratio of cell surface signal to GFP intensity was used for normalization. The mean ± SEM of three experiments is shown. End-point measurement (2 hours) of the effects of the hit molecules on the trafficking of CCR5 (B), CCR1 (C), and CXCR4 (D) in HeLa cells. Cells were pretreated for 1.5 hours with molecules at 10 μM. Cargo present at the cell surface 2 hours after release from the ER was measured by flow cytometry using an anti-GFP antibody. Ratio of cell surface signal to GFP intensity was used for normalization. The mean ± SEM of three experiments is shown. Real-time synchronized secretion of CCR5 and CCR1 was monitored using dual-color imaging in nontreated HeLa cells (E) and in HeLa cells pretreated for 1.5 hours with 10 μM molecule 13 (F). Scale bars, 10 μm.

  • Fig. 4 Molecules 13 and 14 inhibit CCR5 secretion via its cytoplasmic tail and inhibit palmitoylation.

    (A) Schematic representation of CCR5 with three palmitoylated cysteine residues indicated in blue. (B) Amino acid sequence of CCR5, CCR1, their chimeras, and the cysteine to alanine mutants used in this study. (C) Kinetics of the synchronized transport of CCR5/CCR1 chimeras to the cell surface measured by flow cytometry in HeLa cells transiently expressing the constructs. Release was induced by addition of biotin at time 0. The mean ± SEM of three experiments is shown. (D) End-point measurement (2 hours) by flow cytometry of the effects of molecules 13, 14, and 15 on the transport of the CCR5/CCR1 chimeras in HeLa cells after transient expression. Cells were pretreated for 1.5 hours with molecules at 10 μM. The mean ± SEM of three experiments is shown. (E) Kinetics of the synchronized transport of CCR5 cysteine mutants to the cell surface measured by flow cytometry in HeLa cells transiently expressing the corresponding constructs. Release was induced by addition of biotin at time 0. The mean ± SEM of three experiments is shown. (F) End-point measurement (2 hours) by flow cytometry of the effects of molecules 13, 14, and 15 on the transport of CCR5 cysteine mutants. HeLa cells transiently expressing the cysteine mutants were pretreated for 1.5 hours with molecules at 10 μM. The mean ± SEM of three experiments is shown. (G and H) Quantification of palmitoylation of either GFP-CCR5 or GFP-CCR5 Cys3A transiently expressed in HEK293T cells. [3H]Palmitate was incorporated for 4 hours in the presence of the compounds following a pretreatment with DMSO (−) and molecules 13, 14, and 15 for 30 min. A representative autoradiogram and immunoblot are shown (G), and the mean ± SEM of four experiments is shown.

  • Fig. 5 Treatment with molecules 13, 14, and 15 decreases HIV-1 R5 infection in human macrophages.

    Primary human macrophages differentiated for 4 days with rhM-CSF were treated during 18 hours with molecule 13 at 10 μM, molecule 14 at 3 μM, molecule 15 at 1 μM, and molecules 14 and 15 at 1 μM (or DMSO at 0.1%). (A) Cell surface expression of CCR5 was measured by flow cytometry with specific antibodies. (B) Principles of the HIV-1 entry test used (22). Inhibition of fusion of HIV-1ADA (R5 tropic strain) (C) or HIV-1VSVG (VSVG pseudotyped) (D) containing BlaM-Vpr (BV) with CCF2/AM primary macrophages mediated by compounds. Total p24 amount (supernatants and lysates) of HIV-1ADA–infected macrophages was measured by ELISA (E). Each black point represents one donor analyzed independently. Statistical analyses were performed using Prism software. A one-sample t test was applied, and significant P values (<0.05) are indicated for each treatment compared to DMSO in (A), (C), and (E). The absence of a P value indicates that the results were not significantly different. Error bars correspond to SEM.

  • Table 1 Names and molecular formulas of the molecules.

    #MoleculeMolecular formulaLibrary
    1EtretinateC23H30O3Prestwick
    2Pimethixene maleateC23H23NO4SPrestwick
    3NCI 169627C40H49NO14NCI
    4Clemastine fumarateC25H30ClNO5Prestwick
    5CilnidipineC27H28N2O7Prestwick
    6Fluoxetine hydrochlorideC17H19ClF3NOPrestwick
    7Deptropine citrateC29H35NO8Prestwick
    8ParthenolideC15H20O3Prestwick, NCI
    9NCI 1771C6H12N2S4NCI
    10Chlorprothixene hydrochlorideC18H19Cl2NSPrestwick
    11NCI 111118C13H8Cl2S3NCI
    12NCI 228155C11H6N4O4SNCI
    13Cadmium chlorideCdCl2NCI
    14NCI 68093, Zn pyrithioneC10H8N2O2S2ZnNCI
    15NCI 333856, tetrocarcin AC67H96N2O24.NaNCI

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax0821/DC1

    Fig. S1. Molecules 13 and 14 inhibit autopalmitoylation of DHHC3 and DHHC7.

    Fig. S2. Molecules 13, 14, and 15 do not alter CXCR4 surface expression or induce cytotoxicity in primary macrophages.

    Movie S1. Synchronized transport of CCR5.

    Movie S2. Synchronized transport of TNF.

    Movie S3. Synchronized transport of CCR5 and TNF.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Molecules 13 and 14 inhibit autopalmitoylation of DHHC3 and DHHC7.
    • Fig. S2. Molecules 13, 14, and 15 do not alter CXCR4 surface expression or induce cytotoxicity in primary macrophages.
    • Legends for movies S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Synchronized transport of CCR5.
    • Movie S2 (.mov format). Synchronized transport of TNF.
    • Movie S3 (.mov format). Synchronized transport of CCR5 and TNF.

    Files in this Data Supplement:

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