Fig 1: Knockout of the CCL5 receptors CCR3 and CCR5 limits ZIKV infection of hBMECs. (A) Puromycin-selected CCR3-KO and CCR5-KO hBMECs were evaluated for CCR3 or CCR5 protein expression, compared to GAPDH, by Western blotting. (B and C) WT, CRISPR control, and CCL5-KO hBMECs were ZIKV infected (MOI of 10), and ZIKV infection was detected by ZIKV antigen-positive cells at 18 hpi (B) and 3 dpi (C). (D and E) At 3 dpi, supernatants were assessed for viral titers (D), and viability was assessed via CyQuant uptake (E). Asterisks indicate statistical significance (*, P < 0.05; ***, P < 0.001). Experiments were performed at least 3 times with similar results.
Fig 2: CCR3/CCR5 receptor antagonists restrict ZIKV persistence and viral titers in hBMECs. (A and B) hBMECs were ZIKV infected (MOI of 10) and treated with a CCR3 inhibitor (UCB35625) (20 µM), a CCR5 inhibitor (maraviroc; 20 µM), or both inhibitors twice daily to 3 dpi. The number of ZIKV-infected hBMECs was assessed at 3 dpi (A), and protein levels of ZIKV NS5 and ZIKV Env were quantified compared to GAPDH controls (B). (C and -D) Mock- and ZIKV-infected hBMECs were infected (MOI of 5) and treated with CCR3 or CCR5 inhibitors at 1 to 80 µM concentrations twice daily to 0 to 3 dpi. Treated hBMECs were quantified for the number of ZIKV-infected cells (C) and viral titers (D). Experiments were performed at least 3 times with similar results.
Fig 3: CCL5-CCR3/5 neutralization decreases ZIKV infection in hBMECs. hBMECs were infected with ZIKV (MOI of 5) and treated with isotype control or CCL5-, CCR3-, or CCR5-neutralizing antibodies (10× antibody ND50) added to supernatants once daily. (A) hBMECs were starved overnight at 3 dpi, and ZIKV NS5 was quantified by Western blot analysis and normalized to GAPDH levels. (B) ZIKV antigen-positive hBMECs were detected by anti-DENV4 hyperimmune mouse ascitic fluid (HMAF) and quantified at 3 dpi. (C) Viral titers of ZIKV-infected hBMEC supernatants were determined by a focus-forming unit (FFU) assay at 3 dpi. (D) At 9 dpi, daily neutralizing antibody-treated ZIKV-infected hBMECs were assessed for ZIKV antigen-positive hBMECs. Asterisks indicate statistical significance (*, P < 0.05; ***, P < 0.001). Experiments were performed at least 3 times with similar results.
Fig 4: Exogenous CCL5 rescues the viability and persistence of ZIKV-infected CCL5-KO hBMECs. (A to D) CCL5-KO hBMECs were mock or ZIKV infected (MOI of 10) and control treated with PBS or CCL5 (100 ng/ml) once daily or twice daily to 3 dpi. At 3 dpi, CCL5-KO hBMECs were assessed for viability via calcein-AM/propidium iodide staining (A) and quantified by CyQuant analysis (B) for ZIKV-infected hBMECs (C) and viral titers (D). (E) Lysates of CCL5-KO hBMECs treated with PBS or CCL5 twice daily for 1 and 3 dpi were assessed for ZIKV (ZV) Env, compared to GAPDH controls. (F) WT, CRISPR control, CCL5-KO, CCR3-KO, and CCR5-KO hBMECs were ZIKV infected as described above; control treated with PBS or CCL5 (100 ng/ml) twice a day for 3 dpi; and assessed for ZIKV infection. Asterisks indicate statistical significance (*, P < 0.05; ***, P < 0.001). Experiments were performed at least 3 times with similar results.
Supplier Page from Thermo Fisher Scientific for CCR5 Antibody