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. 2014 Dec 16;89(1):165–180. doi: 10.1128/JVI.01677-14

FIG 1.

FIG 1

Setting up an in vitro HCV rolling-circle replication system. (A) Purification of HCV proteins and RPA. HCV proteins NS3-4A, NS3 helicase, NS5A(S25-C447), NS5A(S25-K215), NS5BΔ21 polymerase and the native human RPA were purified as described in Materials and Methods and analyzed using SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. The arrowheads indicate the bands corresponding to individual proteins or protein subunits in the case of NS3-4A and RPA. (B) Design of the rolling-circle RNA template. Two RNA oligonucleotides (74 nt and 106 nt) were synthesized with T7 RNA polymerase on the primed DNA templates with T7 promoter sequence primers as described in Materials and Methods. The 74-nt RNA oligonucleotide was hybridized with the DNA oligonucleotide “bridge” and ligated with DNA ligase to create a RNA circle. The rolling-circle RNA template was constructed by hybridization of the RNA circle and the 106-nt RNA oligonucleotide to complete the formation of the rolling-circle RNA template.