High titres of recombinant adenovirions encoding the HCV proteins (rAdHCV-CE1E2) were produced in 293T cells by serial passaging and the equivalent multiplicity of infection (MOI) was determined as described previously [24] . To produce HCV VLPs, Huh7 cells were infected with rAdHCV-CE1E2 at a MOI of 1. At 72 hours post-infection, cells were collected and disrupted using a dounce homogeniser and centrifuged at 17,000 g for 5 min. The supernatant was further centrifuged through a 30% sucrose cushion (containing 20 mM Tris pH 7.4 and 150 mM NaCl) at 178,000 g for 4 hours at 4uC. The resulting pellet was resuspended in 50 mM Tris pH 7.and 100 mM NaCl and purified through a 33% caesium chloride gradient by ultracentrifugation at 14uC at 143,000 g) for 72 hours. Twelve 1 ml gradients were recovered and dialysed against sterile PBS at 4uC overnight. Fluorescent labelling of VLPs was achieved by adding VLPs (6 mg/ml) to 2 mg/ml of fluorescein isothiocyanate (FITC; Sigma Aldrich) in 50 ml of DMSO. The suspension was vortexed vigorously and incubated overnight at 4uC before dialysis against PBS the next day. All VLP preparations were stored in aliquots at 270uC until use. Huh7 and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen USA) supplemented with 10% fetal calf serum (FCS) and streptomycin 50 mg/ml at 37 C in 5% CO 2 .