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Structural insights into hepatitis C virus receptor binding and entry


Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2–2A12–CD81-LEL); E2 in complex with 2A12 (E2–2A12); and CD81-LEL alone. After binding CD81, residues 418–422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520–539. Docking of the E2–CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.

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Fig. 1: Ribbon diagrams of the tCD81-LEL–eE2(ΔHVR1)–2A12, eE2–2A12 and tCD81-LEL X-ray crystal structures.
Fig. 2: Ribbon diagrams of the conformational variation in E2 and CD81-LEL.
Fig. 3: Neutralizing antibodies compete directly with CD81 for E2 binding.
Fig. 4: Proximity and interaction of eE2 with membranes.

Data availability

The coordinates and structure factors for eE2–2A12, tCD81-LEL and tCD81-LEL–eE2(ΔHVR1)–2A12 have been deposited into the RCSB PDB ( under accession numbers 7MWW, 7MWS and 7MWX, respectively.


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We acknowledge A. Khan, M. Miller,M. Paskel and L. Tuberty for technical assistance; D. Wu and G. Piszczek for help with the ITC measurements; C. Rice for reagents and advice; and F. Jiang for his dedication to science and friendship. This work was supported by the Intramural Research Programs of the National Institute of Allergy and Infectious Diseases (J.I.C. and J.M.) and NIH grants R01AI136533, R01AI124680, R01AI126890 and U19AI159819 to A.G. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract numbers W-31-109-Eng-38 (SER-CAT) and DE-AC02-06CH11357 (LRL-CAT). SER-CAT is supported by its member institutions, and equipment grants (S10_RR25528 and S10_RR028976) from the National Institutes of Health.

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Authors and Affiliations



A.K., R.A.H., S.A.Y. and Y.W. purified the proteins and determined crystallization conditions. A.K., R.A.H., S.A.Y., W.B., A.D.D., J.I.C. and J.M. collected, processed and analysed the results. A.G. provided the antibody hybridoma. All authors helped to write and edit the manuscript.

Corresponding author

Correspondence to Joseph Marcotrigiano.

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Competing interests

A.K., W.B., A.D., J.I.C, and J.M., are named as inventors on a patent application describing the data presented in this paper, which has been filed by the National Institutes of Health.

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Peer review information Nature thanks Yorgo Modis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Sequence divergence between human and tamarin CD81.

a, Sequence alignment (light blue and black font) of full-length human and tamarin CD81 (Accession numbers: Human NM_004356, Tamarin CAB89875.1). The CD81-LEL (black font) has five divergent residues (green and yellow highlights represent nonidentical and similar amino acids, respectively). b, Ribbon diagram of tamarin CD81-LEL (blue) bound to eE2(ΔHVR1) (red and CD81-binding loop green) with side chains of the five, diverging CD81 residues (blue sticks) and proximal residues in eE2 (red sticks).

Extended Data Fig. 2 Thermodynamic characterization of tamarin and human CD81-LEL interaction with eE2.

ad, ITC for the titration of tCD81-LEL (a, b) or hCD81-LEL (c, d) into eE2 at pH 7.5 (a, c) and pH 5.0 (b, d). Thermogram (upper panel), integrated heats and error bars (middle panel), and fit residuals (lower panel) are shown for each. The measurements were performed at 20 °C and analysed with an A + B AB heterodimer model. Error bars indicate the error of peak integration over an interpolated baseline with a 68% (1 sigma) confidence interval. Residuals are the y-axis difference between the data point and the fitted curve in kcal mol−1.

Extended Data Fig. 3 The asymmetric unit for the tCD81-LEL–eE2(ΔHVR1)–2A12 complex.

a, b, eE2(ΔHVR1) chains C and G (red with extended CD81-binding loop in green), tCD81-LEL chains D and H (blue), 2A12 (wheat) ribbon diagrams in the asymmetric unit of the tCD81-LEL–eE2(ΔHVR1)–2A12 complex from side (a) and top (b) views. The 90° axis of rotation is indicated. Carbohydrate moieties (yellow heteroatom sticks) are also shown.

Extended Data Fig. 4 Diagram and conservation of HCV E2.

a, Schematic representation of the E2 protein with the CD81-binding loop highlighted in yellow and the asterisks highlighting regions associated with CD81 binding. b, Multiple sequence alignment of eE2 from representative strains (as labelled) of the seven genotypes. Conserved residues (cyan highlights) and CD81-binding loop residues (red font) are noted. Asterisks indicate residues ≤4Å from tamarin CD81 common to both chains C and G (red), chain G only (blue), and chain A only (green). Hypervariable region, antigenic site, and transmembrane are labelled HVR, AS and TM, respectively.

Extended Data Fig. 5 A simulated-annealing 2FoFc composite omit map for the eE2(ΔHVR1) CD81-binding loop in the X-ray crystal structure of the complex.

a, b, CD81-binding loop in (a) Chain C and (b) Chain G (green heteroatom sticks), residues as labelled, in a 0.8σ contour level 2FoFc composite omit map (blue mesh) calculated from the omission of residues 415–426 and 520–539, and packed against the tCD81-LEL (blue) and eE2(ΔHVR1) (red) ribbon diagrams.

Extended Data Fig. 6 Interface between tCD81-LEL and eE2(ΔHVR1).

Ribbon diagram of tCD81-LEL (blue) and eE2(ΔHVR1) (red) interface, chains C and D, with side chains (blue and red heteroatom sticks, respectively). The labels for tCD81-LEL residues are underlined.

Extended Data Fig. 7 Electrostatic-potential surface maps of E2 and tCD81-LEL.

aj, Electrostatic-potential surface maps of eE2(ΔHVR1) in complex (a, b), tCD81-LEL–eE2(ΔHVR1) complex (c, d), tCD81-LEL in complex (e, f) and free form (g, h), and full-length eE2 free form (i, j). The surfaces are coloured by electrostatic potential corresponding to +5 kcal/(mol·e) (blue) and −5 kcal/(mol·e) (red) at 298 K calculated at pH 7.5 (a, c, e, g, i) and 5.0 (b, d, f, h, j) as labelled. Panels a, b, i, and j are depicted in the same orientation; panels eh are depicted in the same orientation. a, b, tCD81-LEL is shown as a transparent, blue ribbon diagram. e, f, The eE2(ΔHVR1)-binding surface is outlined with a dotted line.

Extended Data Fig. 8 Expression, purification and liposome flotation of eE2 mutants.

a, E2-specific western blot of cell culture supernatants showing secreted protein levels of eE2 mutants I422A, Y529A, W531A, and double mutant Y529A/W531A. Expi293 GnTI cells were transfected and supernatants (uncleaved eE2 protein) were mixed with reduced 2x sample buffer. 15 ul of supernatant was loaded in each well. E2 2C1 primary antibody was used for western blotting. b, Coomassie-stained 4-20% Bis-Tris SDS-PAGE gels of purified eE2 mutant proteins in the presence (Reduced) and absence (Non-reduced) of β-mercaptoethanol. c, E2-specific western blot of top fractions from liposome flotation assays, comparing increased loading (as labelled under each blot) of mutants. Protein molecular weight maker (L) and wild-type eE2 is provided as a marker (std). Sample pH, inclusion of tCD81-LEL, and eE2 mutant proteins are labelled.

Extended Data Table 1 Affinity measurements of eE2 to human and tamarin CD81-LEL at neutral and low pH
Extended Data Table 2 Data collection and refinement statistics for eE2–2A12, tCD81-LEL and tCD81-LEL–eE2(ΔHVR1)–2A12 complexes
Extended Data Table 3 Residues making interactions within ≤4 Å

Supplementary information

Supplementary Figure 1

This file contains the uncropped gel source data shown in Fig. 4c.

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Kumar, A., Hossain, R.A., Yost, S.A. et al. Structural insights into hepatitis C virus receptor binding and entry. Nature 598, 521–525 (2021).

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