HIV-1 infection assays used viruses derived from the infectious molecular clones pNL4.3-BaL.ecto (R5-tropic) and pNL4.3 (X4tropic), supplied by John Kappes and Christina Jambor (University of Alabama at Birmingham, USA). Cryopreserved healthy donor PBMCs were stimulated with 2.5ug ml 21 PHA (Sigma Aldrich, St. Louis, MO) for 2 d in R10 medium (Invitrogen, Carlsbad, CA) and 1 d with 50U ml2 1 IL-2 (Roche, Nutley, NJ) in R10 medium adjusted to contain 20% serum. MDDCs were generated by culturing CD14 + cells isolated from buffy coats (National Blood Service, London, UK) using a Human Monocyte Isolation Kit II medium was never more than 0.2%. Control cultures were treated with a similar volume of 2% DMSO OptiMEM or plain OptiMEM (vehicle only) to match the addition of inhibitor. PBMCs were preincubated with inhibitors for 2 h prior to infection, then were infected with R5 or X4 virus at a MOI of 0.01 for 16 h at 37uC in the presence of inhibitor. Excess virus and inhibitor were washed out and cells were cultured for 7 d in R10 medium plus 50U ml 21 IL-2. Supernatants were harvested and p24 levels were determined by ELISA (Advanced BioSciences Laboratories, Inc., Kensington, MD). MDDCs were preincubated with inhibitors for 1 h prior to infection at a MOI of 0.1 for 2 h at 37uC in the presence of inhibitor; and after washout of inhibitor and excess virus, cells were cultured for 4 before reading out supernatant p24 levels as described above. Statistical analysis of the data from the viral inhibition assays was performed using a one-way ANOVA.