FIG. 1.

Identification of amino acids in the NS4B C terminus that are critical for HCV RNA replication. (A) Schematic representation of the predicted topological arrangement of NS4B bound to the ER membrane. NS4B is predicted to contain four TMDs (numbered 1 to 4) that span the ER membrane, with the flanking N- and C-terminal ends located in the cytosol. Previously predicted features (α-helix between amino acids 6 and 29 and TMX) in the N-terminal region and the location of a nucleotide binding motif (NBM) are shown. Helices (H1 and H2), predicted by PSIPRED, that are located in the C-terminal domain are indicated. Below the cartoon, the amino acid sequence for the C-terminal region of NS4B encoded by strain JFH1 is presented. Amino acid residues that were replaced with alanine are underlined and numbered M1 to M15. Amino acids predicted to lie within helices H1 and H2 are overlined and shown in red. Asterisks denote conserved amino acids, and residues are numbered according to the N-terminal end of NS4B following cleavage from the polyprotein by the NS3/4A protease. (B) Schematic representation showing the Luc-JFH1_GFP SGR encoding NS5A-GFP and the position of the C-terminal end of NS4B containing mutations M1 to M15. (C) Huh-7 cells were electroporated with in vitro-transcribed RNA derived from wt Luc-JFH1_GFP, mutants M1_luc to M15_luc, and Luc-JFH1_GND. Extracts were prepared at 4, 24, 48, and 72 h to determine luciferase activity at each time point. Assays were performed in duplicate. RLU, relative light units.