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

Khan AG, Whidby J, Miller MT, Scarborough H, Zatorski AV, Cygan A, Price AA, Yost SA, Bohannon CD, Jacob J, Grakoui A, Marcotrigiano J - Nature (2014)

Bottom Line: 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.These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. 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 antibodies. 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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Overview of HCV E2a, Schematic representation of the HCV genome and E2 domain organization. Full-length eE2 and the crystallization construct are indicated by the black and grey bars, respectively. C, capsid protein; nonstructural protein (NS) 2-5B. Stars indicate the location of trypsin (blue), chymotrypsin (green) and GluC (magenta) cleavage sites. Ribbon diagram of the E2 core domain bound to Fab 2A12 (b) and alone (c and d). The view in d is a 90° rotation about a horizontal axis from c. The E2 polypeptide chain is colored from the N terminus (blue) to C terminus (red). e, Topology diagram of E2 core domain, detailing secondary structure elements, disulfide bonds (dashed lines), N-linked glycosylation sites and regions of disordered polypeptide (dotted lines).
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Figure 8: Overview of HCV E2a, Schematic representation of the HCV genome and E2 domain organization. Full-length eE2 and the crystallization construct are indicated by the black and grey bars, respectively. C, capsid protein; nonstructural protein (NS) 2-5B. Stars indicate the location of trypsin (blue), chymotrypsin (green) and GluC (magenta) cleavage sites. Ribbon diagram of the E2 core domain bound to Fab 2A12 (b) and alone (c and d). The view in d is a 90° rotation about a horizontal axis from c. The E2 polypeptide chain is colored from the N terminus (blue) to C terminus (red). e, Topology diagram of E2 core domain, detailing secondary structure elements, disulfide bonds (dashed lines), N-linked glycosylation sites and regions of disordered polypeptide (dotted lines).

Mentions: HCV envelope glycoprotein 2 (E2) is a type II transmembrane protein with an amino-terminal ectodomain connected to a carboxyl-terminal transmembrane helix through an amphipathic, alpha helical stem (Fig. 1a) 5,6. E2 is highly modified post translationlly with 9 to 11 N-linked glycosylation sites and 18 cysteine residues that are conserved across all genotypes. For ease of comparison with other genotypes, the cysteines and N-linked glycosylation sites will be referred to as C1 to C18 and N1 to N11, respectively, with residue numbers from the J6 (2a) genome given in parentheses. Full-length, E2 ectodomain (eE2) (384-656) was produced in HEK293T GnTI- cells by a lentiviral expression system and grown in an adherent cell bioreactor. The resulting eE2 protein is monomeric as determined by non-reducing SDS-PAGE and size exclusion chromatography (SEC) (Extended Data Fig. 1).


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

Khan AG, Whidby J, Miller MT, Scarborough H, Zatorski AV, Cygan A, Price AA, Yost SA, Bohannon CD, Jacob J, Grakoui A, Marcotrigiano J - Nature (2014)

Overview of HCV E2a, Schematic representation of the HCV genome and E2 domain organization. Full-length eE2 and the crystallization construct are indicated by the black and grey bars, respectively. C, capsid protein; nonstructural protein (NS) 2-5B. Stars indicate the location of trypsin (blue), chymotrypsin (green) and GluC (magenta) cleavage sites. Ribbon diagram of the E2 core domain bound to Fab 2A12 (b) and alone (c and d). The view in d is a 90° rotation about a horizontal axis from c. The E2 polypeptide chain is colored from the N terminus (blue) to C terminus (red). e, Topology diagram of E2 core domain, detailing secondary structure elements, disulfide bonds (dashed lines), N-linked glycosylation sites and regions of disordered polypeptide (dotted lines).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126800&req=5

Figure 8: Overview of HCV E2a, Schematic representation of the HCV genome and E2 domain organization. Full-length eE2 and the crystallization construct are indicated by the black and grey bars, respectively. C, capsid protein; nonstructural protein (NS) 2-5B. Stars indicate the location of trypsin (blue), chymotrypsin (green) and GluC (magenta) cleavage sites. Ribbon diagram of the E2 core domain bound to Fab 2A12 (b) and alone (c and d). The view in d is a 90° rotation about a horizontal axis from c. The E2 polypeptide chain is colored from the N terminus (blue) to C terminus (red). e, Topology diagram of E2 core domain, detailing secondary structure elements, disulfide bonds (dashed lines), N-linked glycosylation sites and regions of disordered polypeptide (dotted lines).
Mentions: HCV envelope glycoprotein 2 (E2) is a type II transmembrane protein with an amino-terminal ectodomain connected to a carboxyl-terminal transmembrane helix through an amphipathic, alpha helical stem (Fig. 1a) 5,6. E2 is highly modified post translationlly with 9 to 11 N-linked glycosylation sites and 18 cysteine residues that are conserved across all genotypes. For ease of comparison with other genotypes, the cysteines and N-linked glycosylation sites will be referred to as C1 to C18 and N1 to N11, respectively, with residue numbers from the J6 (2a) genome given in parentheses. Full-length, E2 ectodomain (eE2) (384-656) was produced in HEK293T GnTI- cells by a lentiviral expression system and grown in an adherent cell bioreactor. The resulting eE2 protein is monomeric as determined by non-reducing SDS-PAGE and size exclusion chromatography (SEC) (Extended Data Fig. 1).

Bottom Line: 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.These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. 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 antibodies. 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.

Show MeSH
Related in: MedlinePlus