Figure 1.
Production and biochemical characterization of HCV E2e.
A) Schematic diagram of the HCV genome region coding for the structural proteins and the constructs used to make stable S2 cell transfectants expressing E2e. MT: inducible metallothionin promoter, BiP: Drosophila BiP Signal peptide, EK: enterokinase cleavage site, ST: Strep-Tag. B) Elution profile of E2e from the HCV isolates indicated from an Sdx200 size exclusion column. Bottom panels: Non-reducing SDS-PAGE analysis of the eluted fractions. Arrows indicate multimeric (A), dimeric (D) and monomeric (M) forms of the protein.
Table 1.
Production yields of E2e constructs from the 9 selected HCV isolates.
Figure 2.
Functional and conformational characterization of HCV E2e.
A) Stoichiometric complex formation between H77 E2e and mAb H53. H77-E2e, mAb H53 and a mixture of the two (molar ratio 2∶1) were loaded to the column (in three different runs) (E2e∼50kD, H53∼150kD, complex∼250kD). No peaks corresponding to either of the isolated proteins were observed in the profile of the complex, indicating a 2∶1 complex stoichiometry and a high affinity of H77 E2e for mAb H53. B) Dose-dependent inhibition of infection of Huh7.5 cells by HCVcc. Huh-7.5 cells were preincubated with increasing concentrations of HCV E2e, WNV sE or BVDV E2e and subsequently infected with HCVcc in the corresponding recombinant protein concentration. The number of infected foci was determined after immunofluorescence analysis detecting intracellular HCV core antigen. The columns represent mean values of duplicates in a representative experiment; bars indicate mean deviation, 100% corresponds to the mean value of the infection in the presence of the control proteins.
Figure 3.
Amino acid sequence alignments and secondary structure predictions.
The secondary structure of HCV E2e and alphavirus sE1 was predicted using the program DSC [48], which was selected because the predictions matched more closely the crystallographically determined secondary structure elements of class II proteins. Arrows or spirals under each sequence indicate predicted β-strands or α-helices, respectively. A) The main elements of the tertiary structure model of HCV E2 are framed: assigned strands in DI (red, labeled) and DIII (blue), putative fusion loop (yellow), the stem (grey) and regions that can be deleted without affecting the protein conformation (brown). The 18 cysteines, which form the 9 disulfides, are marked with arrows and numbered according to the disulfide bond (Table 2) under the sequences. N-linked glycosylation sites are numbered in green. Residues known to interact with CD81 are marked with small blue circles. The numbering corresponds to the HCV H77 polyprotein. B) Comparison with a class II fusion protein of known structure. The experimentally determined secondary structure of SFV E1 taken from the crystal structure (PDB 2ALA, [49]), shown with symbols (arrows or spirals) above the sequence alignment, was compared with secondary structure predictions for the E1 ectodomain of selected alphaviruses. Experimentally determined β-strands in DI, DII and DIII are colored red, black and blue, respectively. The red frames indicate the consensus predicted strands in DI, for easier comparison with panel A. Similarly the fusion loop (yellow), the region corresponding to the crystallographically identified DIII (blue), and the stem (grey) are framed. The 16 cysteines forming the 8 disulfides are numbered in black according to the disulfide bond under the sequences. The numbering starts with the first amino acid of the E1 glycoprotein.
Figure 4.
Experimental analysis of the secondary structure of HCV E2e.
A) Far-UV CD spectra obtained with recombinant HCV E2e (empty triangles) and the controls CHIKV sE1 (grey circles) and WNV sE (black diamonds). The inset shows the estimated fraction of α-helix, β-sheet, turns and unordered polypeptide chain for the 3 proteins. B) FTIR spectra of HCV E2e (dashed black line) and DV3 sE (solid black line) and the difference spectrum of the two after normalization (dashed grey line) in the amide I band region.
Figure 5.
A) Typical HPLC elution profile of a tryptic digest of E2e under non-reducing (black) and reducing (light grey) conditions, superposed to highlight the difference in mobility upon reduction. Asterisks mark peaks that disappeared upon reduction and were thus selected for further proteomic analysis. B) Mass spectrum of a sample recovered from an HPLC peak susceptible to reduction (Peak 16-3 of JFH-1 E2 identified as peptide J4 in the detailed description provided in SI). Upon reduction, a shift in molecular mass of 2Da was observed, due to addition of two hydrogen atoms upon reduction of the two cysteines. The data presented in this figure allowed the identification of disulfide 4 (Table 2).
Table 2.
Recapitulation of the 9 disulfide bridges and the isolates in which they were identified.
Figure 6.
Tertiary organization of HCV E2e.
A) The linear sequence of HCV H77-E2e, numbered every 10 residue according to the polyprotein (N-terminus at position 384 and the TM region beginning at 716) is represented as a chain of beads (colored circles) labeled with the corresponding amino acid and threaded onto a class II fold as described in the text. Circles in pale and bright colors represent residues in the background and foreground of the domains, respectively labeled in white and black fonts. Disulfide bonds and glycosylation sites are indicated by thick black bars and green circles, respectively, numbered sequentially. Unstructured segments are in white font on a brown background. Residues that participate in CD81 binding are contoured in blue, and those from the putative fusion loop region in red. In DIII, a plausible arrangement of the 3 predicted β-strands is illustrated by placing the corresponding polypeptide segments in an antiparallel putative β-sheet, but unlike DI, the topological arrangement of this domain was not determined. B) Schematic diagram of the tertiary organization of HCV E2, with DI, DII and DIII in red, yellow and blue, respectively. The stem region is grey. The connectivity of the β-strands in DI is indicated, labeled with the standard class II nomenclature. A broken yellow line indicates the place of the second insertion in other class II proteins.