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. 2013 Nov 29;342(6162):1090-4.
doi: 10.1126/science.1243876.

Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

Free PMC article

Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

Leopold Kong et al. Science. .
Free PMC article


Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.


Fig. 1
Fig. 1. Structures of HCV E2 and comparison with the class II fusion fold
(A) The crystal structure of HCV E2c is displayed as a cartoon representation and colored by structural components: the front layer is formed by the N-terminal region residues 421–453 (cyan); the outer (purple) and inner (red) sheet form the Ig β-sandwich; the CD81 receptor binding loop is a bilobed structure (blue); a flexible region (white) encompasses variable region 2 (VR2); and the back layer (597–645) is formed by two short helices, loops and a 4-stranded β-sheet (light green). Labeling of β-sandwich strands follows Ig-fold conventions. Disulfide bonds are shown as yellow sticks and numbered from the N-terminus. N-linked glycans are indicated by green circles and are also numbered from the N-terminus. Asterisks indicate N-linked glycans deleted in the construct. Disordered regions in the structure are shown by dotted lines. An inset shows the structure of a loop in the E2c structure from complex B that is disordered in complex A. (B) Topology diagram of E2c following the same coloring scheme as in the cartoon representation. (C) Scaled comparison of HCV E2c (top) and TBEV E protein, the canonical class II fusion protein (PDBID: 1SVB). DI–III indicates domains I–III. (D) The 16 Å EM density map of HCV E2ΔTM bound to Fab AR3C (transparent gray surface) is shown from two perspectives with the crystal structure of HCV E2c bound to Fab AR3C fitted into the EM density. The crystal structure is displayed as a ribbon, with the E2c colored as in Figure 1. Dotted lines indicate the main portions of E2ΔTM that are absent in the E2c crystal structure. The numbers of amino acids (aa) in the missing regions are shown. Blue dotted lines regions in E2ΔTM are not in the E2c construct and black dotted lines indicate regions that are in the E2c construct but are disordered in the crystal structure. Two protrusions in the EM density are in the vicinity of N-linked glycan sites and high mannose glycans (green ball-and-sticks) are modeled at those positions. Measurements of dimensions are rounded to the nearest ten.
Fig. 2
Fig. 2. HCV E2 interaction with Fab AR3C
(A) Overall structure of E2c (red) bound to Fab AR3C is displayed as a cartoon representation with the heavy and light chains of Fab colored dark and light green, respectively. N-linked glycans are shown in a ball-and-stick representation with carbon, oxygen and nitrogen atoms colored yellow, red and blue. (B) Interactions between Fab AR3C and E2c. The CDR loops of AR3C are displayed as thick tubes over the gray molecular surface of E2. A relatively unusual disulfide bond in CDR H3 for known human antibody structures is shown in yellow. The N430 glycan that interacts mainly with the light chain of AR3C is shown in yellow and red. Below the diagram is a table listing the surface areas buried on E2 by the different CDR loops. (C) The AR3C epitope on E2 is shown as a cartoon representation from the same perspective as in (B) and colored according to sequence conservation. Residues that are not buried by AR3C are colored gray. Below is a table listing the fractions of the surface on the E2 core protein with respect to sequence conservation (binned and color coded) that are buried by AR3C.
Fig. 3
Fig. 3. CD81 receptor-binding site
(A) Three potential CD81 binding sites on E2 were indicated from previously published alanine-scanning results (19): a surface on the β-sandwich, the top of the CD81 binding loop, and the front layer. E2 mutations used to evaluate the possible binding sites are shown as circles numbered from the N-terminus. A yellow circle indicates substitution by a bulky amino acid and a green circle indicates the introduction of an N-linked glycosylation site. E2 is depicted as a cartoon within its molecular surface and colored as in Fig. 1A. (B) The substitutions shown in (A) are described and their effects on CD81 and Fab AR3C binding are tabulated. Numbers indicate % binding of CD81 or Fab AR3C to variants relative to wild type E1E2 in an ELISA. Binding is color coded: 0–25% = red, 26–50% = yellow, 51–75% = green, >75% = white. (C) Negative-stain EM reconstructions of deglycosylated E2ΔTM bound to Fabs AR2A and AR3C (left) or bound to Fab AR2A and CD81 dimer (right). The crystal structure of E2c bound to Fab AR3C and a model of Fab AR2A displayed as ribbons are fitted within the electron density. E2c is colored as in Fig. 1. Helices C and D of CD81 are highlighted in yellow because they contain residues important for binding to E2 (34). In the CD81 complex, the density suggests that a CD81 dimer is present as in CD81 crystal structures (35, 36).
Fig. 4
Fig. 4. HCV E2 antigenic surface
The E2c structure is displayed as a molecular surface colored according to different antigenic properties described in the text. Missing HVR1 and VR2 regions are represented as colored ovals and labeled. The mAb binding sites mapped from alanine scanning studies or from crystal structures bound to peptides, as described in the main text, are indicated. On the neutralizing face (right), three antigenic regions are shown: residues 412–423 (red dashed line connecting HVR1 and the rest of E2), the AR3C epitope in the front layer that overlaps with the CD81 binding site (cyan dotted line), and α1 in the front layer (white dotted line). The gray surface is relatively hydrophobic and conserve and may be an occluded zone covered by HVR1 in the full-length E2 protein, as suggested by the EM reconstruction of E2ΔTM (Fig. 1C).

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