LTR, sE2 and E1E2 Transfection of Cell Lines

293T cells were seeded on 96 well plates (1.5x10⁴ cells/well). When confluent, media was removed and 50 µL of Opti-MEM added to each well. Two µL Opti-MEM added per well with the following plasmid quantities: 12, 6 or 1 ng E1E2 or sE2 plasmid (nanogram quantity equalised with pCDNA between conditions), 6 ng LTR and 1 ng Tat. PEI was diluted to 0.14 µg/µl in 200 µL Opti-MEM and 2 µL of the diluted PEI was added to the plasmid mix. The plasmid/PEI mix was incubated at rt for 30 min before being added to cells and incubating for 6 h at 5% CO₂/37°C after which Opti-MEM/plasmid mix was removed and 250 µL DMEM added. Plates were measured for luciferase activity 48 h after transfection as described above. The following genotypes of HCV E1E2 Env were used – 1a, 1b, 2b, 3, 4, 5 and 6 (44, 45). HCV sE2 plasmids containing the gene fragment for the soluble ectodomain of the HCV E2 protein (aa363-661, referenced to the HCV strain H77 polyprotein) were PCR amplified from full length E1/E2 plasmids using genotype-specific primers [primer sequences described previously (45)] and Expand High-Fidelity Polymerase PCR System (Roche). PCR products were sub-cloned into the pCDNA plasmid and possessed an in-frame C-terminal 6xHis tag sequence. The following genotypes of HCV sE2 envelope were used – 1a, 1b, 2b, 4, 5 and 6. No corresponding sE2 genotype 3a plasmid was available.