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).

The C-terminal 23 amino acids of GBV-B NS5B mediate membrane association. (A) Sequence analyses of the GBV-B NS5B C terminus. Amino acids are numbered with respect to the NS5B protein and negatively from its C terminus. The following methods to predict TMDs were combined: HMMTOP (http://www.enzim.hu/hmmtop/), Tmpred (http://www.ch.embnet.org/software/TMPRED_form.html), TMHMM (http://www.cbs.dtu.dk/services/TMHMM), TopPred (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form_toppred), and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/). T, predicted transmembrane amino acid; #, consensus minimum transmembrane segment, considering all prediction methods. (B) Subcellular localization of GFP fusion constructs. Plasmids pCMVGFP, pCMVGFP-GBV-B-NS5B, pCMVGFP-GBV-B-NS5BC31, and pCMVGFP-GBV-B-NS5BΔC23 were transfected into U2-OS cells, followed by fixation and confocal laser scanning microscopy. (C) Membrane flotation analyses. Hypotonic lysates of U-2 OS cells transfected with the constructs above were analyzed by equilibrium centrifugation through 5 to 37.5% (wt/vol) Nycodenz gradients. Fractions were collected from the top and analyzed by immunoblotting using monoclonal antibody JL-8 against GFP (Clontech, Palo Alto, CA). Under these conditions, membranes and associated proteins float to the upper, low-density fractions while soluble and aggregated material remains in the lower, high-density fractions.

Comparison of the TMDs of HCV NS5B, GBV-B NS5B, PV3A, and Cb5. (A) HCV/PV3A and HCV/Cb5 TMD chimeras were designed in analogy to the HCV/GBV-B construct, as detailed in the legend to Fig. 2A. Amino acids are numbered with respect to PV3A and Cb5 proteins. For clarity, the ClustalW sequence comparisons are not reported. (B) Amino acid surface representations. Amino acid color coding and labeling are as described in the legend to Fig. 2E. The structure models of the HCV/PV3A and HCV/Cb5 TMDs were constructed as described in the legend to Fig. 2E. In the case of the HCV/PV3A chimeric protein, segment 16 to 44 of the Pf1 major coat protein (PDB entry 2KLV) and segment 21 to 31 of photosystem I subunit PSAX (PDB entry 1JB0) served as templates. The HCV/Cb5 three-dimensional model is based on comparison with segment 198 to 228 of aquaporin (PDB entry 3LLQ). The N termini of the chimeras were positioned at the same location with respect to the membrane in order to simplify comparisons.