Minicircle Transfection for CHIKV Replication

The minicircle forms of CMV-P1234 and MCC-P1234 were obtained as described [53 (link)]. These minicircles and all plasmid DNAs, used for transfection experiments, were purified using Nucleobond Xtra Midi EF kit (Macherey-Nagel). BHK-21, Huh7, U2OS or BSR cells, grown on 35 mm plates to 90% confluence, were co-transfected with mixtures consisting of 1 μg of plasmid encoding CHIKV replicase and 1 μg of plasmid for expression of corresponding RNA template using Lipofectamine LTX reagent according to the manufacturer's protocols (Invitrogen). COP-5 cells were grown and treated similarly and Lipofectamine 2000 (Invitrogen) was used as transfection reagent. At 18 h post transfection cells were lysed with Renilla luciferase assay lysis buffer (Promega) and Fluc and Gluc activities were analyzed using the Dual-Luciferase Reporter Assay kit and Glomax SIS luminometer (Promega); total amount of protein in cell lysates was measured using Bio-Rad protein assay system according to the manufacturer's protocols. In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega).