FIG. 2.

Chimeric HCV/GBV-B replicon. (A) Design of a chimeric replicon with the catalytic domain of HCV NS5B and the membrane anchor segment of GBV-B NS5B. This design is based on (i) the similarity of amino acid sequences (ClustalW alignment) (asterisk, invariant; colon, highly similar; dot, similar), (ii) the predicted transmembrane segments (see Fig. 1A and reference 7 for GBV-B and HCV, respectively), and (iii) the distribution of charged residues. Amino acids are numbered with respect to HCV and GBV-B NS5B proteins and are numbered negatively from the C terminus of GBV-B NS5B. The box indicates the conserved structural motif, as described in the text. (B) Sequencing of the chimeric HCV/GBV-B replicon. The C-terminal chimeric region is depicted. The first number of the clone designation represents the cell colony and the second the bacterial clone. (C and D) Identification of compensatory amino acid changes in the GBV-B NS5B TMD. (C) C-terminal amino acid sequences of the reengineered replicon constructs. (D) RNA replication of the chimeric constructs. In vitro-transcribed replicon RNA was electroporated into Huh-7.5 cells, followed by G418 selection and colony staining 3 weeks later. Cells (6 × 10⁵, 6 × 10⁴, and 6 × 10³ per 100-mm tissue culture dish) were plated from each electroporation. Results of a representative experiment are shown in the upper panel. The lower panel illustrates the mean colony numbers ± standard deviations (SD) from four independent experiments. (E) Three-dimensional structure models of the TMDs of HCV NS5B, the HCV/GBV-B NS5B chimera, and reengineered mutants. Models were tentatively positioned within a phospholipid bilayer. For HCV NS5B and the HCV/GBV-B chimera, both amino acid surface and ribbon representations are shown to highlight the holes at the TMD surface due to the short side chain residues in the conserved structural motif (boxed in panels A, B and C). For the reengineered mutants, the side chain surfaces of key residues are represented together with the overall ribbon representations. Amino acid numbering of the HCV/GBV-B chimera from the C terminus is according to panels A, B, and C. Residues are colored according to side chain chemical properties: hydrophobic, gray; polar (Ser, Thr, Asn, Gln), yellow; basic (Arg and Lys), blue; acidic (Asp and Glu), red; Cys, green. Glycine and alanine residues within the conserved structural motif are magenta and orange, respectively. Mutated residues at positions −12, −13, and −14 in the HCV/GBV-B chimera are colored accordingly. The structure model of the HCV RdRp TMD was deduced from nuclear magnetic resonance analyses of a synthetic peptide comprising NS5B amino acids 563 to 591 (F. Penin, unpublished data). To construct the model structure of GBV-B NS5B TMD, similar α-helical transmembrane segments of known three-dimensional structure were used as templates. The chimera model was assembled by combining the structures of segment 563 to 569 of HCV NS5B (F. Penin, unpublished data), segment 46 to 72 of F1F0 ATP synthase subunit c (Protein Data Bank [PDB] entry 1C0V), and segment 26 to 48 of phospholamban (PDB entry 2KB7) using the Swiss-PdbViewer program (http://spdbv.vital-it.ch/). The figure was generated from three-dimensional atom coordinates using Visual Molecular Dynamics (http://www.ks.uiuc.edu/Research/vmd) and rendered with POV-Ray (http://www.povray.org).