Fig. 5.

CIRE/mDC-SIGN does not bind pathogens known to interact with hDC-SIGN. CHO cells were co-transfected with the neomycin resistance gene plus the cDNA coding for CIRE/mDC-SIGN, hDC-SIGN or the chimeric molecules (CIRE/mDC-SIGN lectin fused to hDC-SIGN stalk and hDC-SIGN lectin fused to CIRE/mDC-SIGN stalk). Transfectants were stained with 5H10 (anti-CIRE) or AZN-1 (anti-DC-SIGN) and counterstained with anti-rat–PE and anti-mouse–FITC, respectively. The level of fluorescence was determined by flow cytometry. Filled histograph denotes the background staining of control transfectants, lacking the binding site of the primary mAb. Continuous line represents the levels of CIRE/mDC-SIGN (A) and hDC-SIGN (B). Dotted line represents the levels of CIRE/mDC-SIGN-lectin fused to hDC-SIGN stalk (A) and hDC-SIGN lectin fused to CIRE/mDC-SIGN stalk (B). The above expression profiles of the CHO transfectants are representative of the expression pattern seen in the independently executed experiments seen in Fig. 5(C–F). CHO cells expressing CIRE/mDC-SIGN, hDC-SIGN or the chimeric molecules (CIRE/mDC-SIGN lectin fused to hDC-SIGN stalk and hDC-SIGN lectin fused to CIRE/mDC-SIGN stalk) or the neomycin resistance gene were tested for interaction with various pathogens. (C) Leishmania mexicana promastigotes were added at a multiplicity of infection of 5:1. Infection proceeded for 24 h at 33°C, before free parasites were removed, cells fixed in methanol and stained with Giemsa. The percent infected cells or cells with attached parasites were counted. The mean ± SEM of two independent experiments is shown. (D) Lentiviral pseudotypes bearing the EBOV-GP. The indicated CHO cell lines were inoculated with a luciferase reporter virus bearing EBOV-GP and luciferase activities in cell lysates were assessed 3 days after infection. The average of two independent experiments performed in quadruplicates is shown. Error bars indicate SEM. (E) HIV-1. Lectin-mediated HIV transmission was assessed by incubating transfectants with a HIV-1 NL4-3 variant harbouring the luciferase gene in place of nef, washing with culture medium and co-cultivation with receptor-positive target cells. Luciferase activities in cell lysates were determined 3 days after the start of the co-cultivation. The average ± SEM from two independent experiments carried out in quadruplicates is presented. (F) HCMV. Strain AD169 was added and incubated (1 h at 4°C in PBS/0.1% BSA/1 mM CaCl₂/2 mM MgCl₂) and percentage of transfectants to which AD169 attached was determined by FACS analysis using an anti-HCMV gB mAb. The data are presented as the mean ± SEM pooled from two independent experiments.