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Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2

Nature volume 509, pages 381–384 (2014)Cite this article

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Abstract

Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide1. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies2,3,4. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of β-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

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Figure 1: Overview of HCV E2.
Figure 2: Ab initio SAXS envelopes of E2 core, eE2(ΔHVR1) and eE2.
Figure 3: Surface features of E2.

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Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited to the Protein Data Bank under accession code 4NX3.

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Acknowledgements

We acknowledge access to the X25 beamline at NSLS and thank the NSLS staff. NSLS is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. We thank J. Tainer and J. Perry for their support and access to SIBYLS beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory. We thank E. Arnold, J. Bonanno, S. K. Burley, J. Chiu, D. Comoletti, E. Elrod, F. Jiang, S. Khare, P. Lobel, A. Shatkin, A. Stock, J. Shires and A. Thanou for providing helpful comments and assistance. Special thanks to C. M. Rice for providing J6 HCV clone and support. This work was supported by a Yerkes Research Center Base Grant RR-00165 (A.G.) and NIH grants P50 GM103368 (J.M.), R01 AI080659 (J.M.) AI070101 (A.G.) and DK083356 (A.G.). A.G.K. was supported by a grant from the New Jersey Commission on Cancer Research (DFHS13CRP001).

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane West, Piscataway, New Jersey 08854, USA,

    Abdul Ghafoor Khan, Jillian Whidby, Matthew T. Miller, Alexandra V. Zatorski, Alicja Cygan, Samantha A. Yost & Joseph Marcotrigiano

  2. Division of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, 100 Woodruff Circle, Atlanta, Georgia 30322, USA,

    Hannah Scarborough, Aryn A. Price, Caitlin D. Bohannon, Joshy Jacob & Arash Grakoui

  3. Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, 100 Woodruff Circle, Atlanta, Georgia 30322, USA,

    Arash Grakoui

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Contributions

The project was initiated, designed and supervised by A.G. and J.M. A.G.K., J.W., A.V.Z., A.C. and S.A.Y. designed protein constructs and established purification protocols. A.G.K. prepared all protein crystals. The mouse monoclonal antibody was produced by H.S. and A.G., and was sequenced by C.D.B. and J.J. A.A.P. and A.G. performed the virus neutralization and patient sera ELISA. J.M., M.T.M. and A.G.K. collected, processed and analysed the X-ray crystallographic and SAXS data. A.G.K., J.W. and J.M. wrote the paper with contributions from all authors.

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Correspondence to Arash Grakoui or Joseph Marcotrigiano.

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Extended data figures and tables

Extended Data Figure 1 eE2, eE2(ΔHVR1) and E2 core are highly soluble and monomeric in solution.

a, A comparison of proteins under reducing and non-reducing conditions is shown by a 10% SDS–PAGE gel with protein standards (Std). b, Size-exclusion chromatography of eE2, eE2(ΔHVR1) and E2 core proteins on a Superdex200 gel filtration column. The elution positions of the void volume (>200 kDa), albumin (66 kDa) and cytochrome C (12.4 kDa) are indicated. Molecular masses of eE2, eE2(ΔHVR1) and E2 core are ∼46 kDa, ∼42 kDa and ∼32 kDa, respectively.

Extended Data Figure 2 eE2 sequence alignment.

Red bent arrows indicate the N- and C-terminal boundaries of the E2 crystallization construct. Cylinders and arrows represent α-helices and β-strands, respectively, and are coloured according to cartoon representation in Fig. 1. CD81 binding regions are bracketed in red; hypervariable regions are bracketed in black. SR-BI binds to HVR1. The asterisks indicate the location of trypsin (blue), chymotrypsin (green) and GluC (magenta) cleavage sites. The binding sites of neutralizing antibodies for which structural information is available are coloured orange for HCV1 and AP33, blue for mAb 8, and purple for HC34-1 and HC34-17.

Extended Data Figure 3 Hydrogen deuterium exchange and limited proteolysis of eE2.

a, The percentage hydrogen deuterium exchange shown at 10, 100 and 1,000 s time points. The secondary structure of E2 core is placed above to emphasize flexible regions. A red arrow indicates the E2 core N terminus. Extra residues (grey) on N and C terminus come from the vector. Potential cleavage sites for trypsin (blue), chymotrypsin (green) and GluC (magenta) are indicated by asterisks. The colour pattern indicates the percentage of exchange. Grey areas are the regions of no coverage. b, Digestion of deglycosylated eE2 with chymotrypsin (left) and GluC (right) reveals a shift from the ∼35-kDa untreated protein (0 min) to ∼25 kDa after digestion. Samples were taken at the indicated time points and analysed by reducing 12% SDS–PAGE gel. Molecular mass protein standards (Std) are indicated. The bands were analysed by N-terminal sequencing and mass spectrometry.

Extended Data Figure 4 Functional analyses of eE2 and E2 core.

a, Antibodies from patient sera infected with HCV genotype 2 show a concentration-dependent binding to eE2 (red) whereas healthy donor sera exhibit only background binding (black). b, Similar binding is observed for E2 core. The measurements were done in triplicate with the error bars representing the standard error of the mean (s.e.m.). c, E2 core (light grey) shows reduced binding to CD81 when compared to eE2 (dark grey) by an ELISA. Bars with stripes indicate E2 binding to a negative control, BSA. The solid black bar indicates CD81 binding to PBS, used to verify the absence of background. The measurements were done in triplicate with the error bars representing the s.e.m. d, eE2 (blue) and CD81-LEL (positive control, grey) inhibit the infection. E2 core (red) shows reduced inhibition. HIV gp140 (black) expressed in the same system was used as a negative control. The measurements were done in triplicate with the error bars representing the s.e.m. e, To rule out the possibility of toxic effects from the recombinant proteins, the cell viability was measured as described in Methods, using similar protein concentrations as in d. f, In an ELISA, 2A12 (red), and an irrelevant antibody, H113 (grey), fail to neutralize HCVcc infection. 2C1 (positive control, black), a mouse monoclonal antibody that binds to the disordered N-terminal region of eE2, blocks infection. The measurements were done in triplicate with the error bars representing the s.e.m.

Extended Data Figure 5 E2 core contains an extensive hydrophobic core.

Sheets A and B are held together by an extensive hydrophobic core composed of mostly aromatic amino acids (green) and five disulphide bonds (yellow).

Extended Data Figure 6 eE2 and E2 core do not undergo oligomeric changes at low pH.

a, b, An overlay of E2 core (a) and eE2 (b) elution profiles from Superdex200 gel filtration at pH 7.5 (blue) and pH 5.0 (red). The expected void volume and observed elution positions of individual proteins are indicated. c, The SAXS envelope of CD81-LEL fit with a dimer crystal structure (PDB 1G8Q). The individual proteins of the CD81-LEL dimer are coloured red and blue.

Extended Data Figure 7 Epitope mapping of conformational antibodies on E2 core surface.

a, Surface epitopes of AR1 (orange) are shown. AR1 blocks the E1E2 heterodimer binding to CD81. AR5A (purple) inhibits E1E2 heterodimerization and is mapped on a well-conserved hydrophobic surface of the core. b, Surface of E2 core coloured by electrostatic potential. The view in a and b is identical.

Extended Data Table 1 Summary of the X-ray crystallographic analyses
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Extended Data Table 2 Summary of SAXS analyses
Full size table

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Khan, A., Whidby, J., Miller, M. et al. Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2. Nature 509, 381–384 (2014). https://doi.org/10.1038/nature13117

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