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. 2016 Nov 10;97(11):2883–2893. doi: 10.1099/jgv.0.000608

Fig. 1.

Fig. 1.

Cloning strategy to generate HCVcc and HCVpp with matched E1E2 proteins. Using PCR and In-Fusion cloning, the H77 E1E2 gene sequence was deleted from the H77/JFH-1 chimeric full-length replication-competent HCV (HCVcc) genome, and an AfeI restriction site was introduced between the Core and P7 genes. This plasmid was then linearized by AfeI digestion and various E1E2 genes from naturally circulating HCVs inserted, generating chimeric H77/natural isolate E1E2/JFH-1 HCVcc genomes. RNA was produced via in vitro transcription and transfected into Huh7.5.1 cells to generate replication-competent virus. Matching natural isolate E1E2 gene sequences were also cloned into the expression vector pcDNA3.2, and this plasmid was co-transfected with the lentiviral luciferase reporter plasmid pNL4-3.Luc.R−E− to produce HCVpp. Colours in the HCVcc genome indicate the HCV variant from which each segment is derived (red, JFH-1; blue, H77; orange, naturally circulating HCV sequence).