Fig 1.

HCV tolerates an HA tag insertion into the N-terminal region of NS4B. (A) Schematic representation of the bicistronic HCV subgenomic reporter replicon. Firefly luciferase (Fluc) or neomycin-phosphotransferase (neo^R) is expressed as an N-terminal fusion with 16 amino acids of the N-terminal region of the core protein (black line) and is translated under the control of the HCV IRES contained in the 5′ UTR. The second cistron (NS3 to NS5B) is translated via the IRES of the encephalomyocarditis virus (EMCV-IRES). (B) Predicted NS4B membrane topology. N-terminal amphipathic α-helices AH1 and AH2, the four transmembrane segments (TM1-4), and the C-terminal α-helices H1 and H2 are schematically depicted. Numbers indicate amino acid positions of the JFH1 isolate. Affinity-tag insertion sites are highlighted by black arrows. (C) Huh7-Lunet cells were transfected with the in vitro-transcribed luciferase replicon RNAs specified at the bottom. Cells were lysed 4, 24, 48, and 72 h after transfection, and the luciferase activity in cell lysates was determined. Data were normalized to the 4-h value that reflects transfection efficiency. The background of the assay is determined by the NS5B active-site polymerase mutant (ΔGDD) (dashed line). Mean values of two independent experiments are shown. Error bars indicate standard deviations. (D) Huh7-Lunet cells were transfected with the in vitro-transcribed luciferase replicon RNAs specified at the bottom. Replication efficiency was determined as described for panel C. (E) Release kinetics of infectious HCV particles. Huh7-Lunet cells were transfected with the full-length HCV RNAs specified on the right. Culture supernatants were harvested at the given time points. Infectivity titers were determined by limiting-dilution assay and are expressed as 50% tissue culture infective doses (TCID₅₀)/ml. Mean values of two independent experiments are shown; error bars indicate standard deviations.