Site-directed mutagenesis was carried out using pcDNA3-DC-SIGN vectors obtained from Drs. S. Pöhlmann, F. Baribaud, F. Kirchhoff and R.W. Doms [26] through the AIDS Research and Reference Reagent Program, NIAID, NIH: pcDNA3-DC-SIGN exon 4 was amplified from genomic DNA of variants carriers and replaced between PspI and EspI (Fermantas, Burlington, Canada) restriction sites. All recombinants clones were verified by DNA sequencing for the presence of mutations of interest and conservation of the coding frame. Stably transfected cell lines were generated from Raji cells (ATCC, Manassas, USA) by nucleofection (Cell line Nucleofector Kit V, Amaxa, Walkersville, USA) and maintained in RPMI 1640 10% FBS containing 1 mg/ ml of G418 (Invitrogen). DC-SIGN-expressing cells were sorted (sorter BD ARIA, BD Biosciences, Mississauga, Canada) and limiting dilutions were performed. Cell lines were grown from a single clone. 3610 5 cells/well of each Raji transfectants were plated in 96-well plates and pre-incubated with mannan (200 mg/ ml, Sigma-Aldrich, St-Louis, USA), DC-SIGN blocking antibody (clone AZN-D1, R&D systems, Minneapolis, USA) (20 mg/ml), matching isotype control or medium for 30 minutes at 4uC before adding 50 ng of p24 equivalent of virus (HIV-1 HxBru-ADA or HIV-1 JRCSF ). Cells were incubated 2 h at 37uC and washed with PBS (Invitrogen). Cells were lysed and assayed for p24 Ag using ELISA. In co-culture experiment, Raji transfectants were pulsed as above and 3610 5 phytohemagglutinin-L activated human primary CD4 + T-lymphocytes (ratio 1:1) isolated from peripheral blood mononuclear cells were added. Cells were cultivated in RPMI 10% FBS 100 U/ml rIL-2 for 5 days. Supernatants were collected and p24 was measured using ELISA. CD4 + T-lymphocytes were isolated from healthy donors and activated as previously described [27] . HIV-1 stocks were generated by transient transfection of HEK293T cells with HxBRU ADA encoding the R5-tropic HIV-1ADA Env [28] or JRCSF proviral construct using the standard calcium-phosphate method. Viral stock was titrated using ELISA p24 Ag (BioChain, Hayward, USA).