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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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Surface features of E2The surface of the E2 core domain colored for (a and d) electrostatic-1 potential: blue (basic), white (neutral), red (acidic) at ± 5 kTe-1 and (b and e) sequence identity (green) from the alignment in Extended Data Fig. 2. Ribbon diagram highlighting the location of the N-linked glycosylation sites are shown (c and f). The orientation of d, e, and f as well as a, b, and c are identical. The orientation in d, e, and f is rotated 180° about a horizontal axis from the view in a, b and c.
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Figure 10: Surface features of E2The surface of the E2 core domain colored for (a and d) electrostatic-1 potential: blue (basic), white (neutral), red (acidic) at ± 5 kTe-1 and (b and e) sequence identity (green) from the alignment in Extended Data Fig. 2. Ribbon diagram highlighting the location of the N-linked glycosylation sites are shown (c and f). The orientation of d, e, and f as well as a, b, and c are identical. The orientation in d, e, and f is rotated 180° about a horizontal axis from the view in a, b and c.

Mentions: HCV E2 is modified by N-linked glycosylation, which is necessary for proper folding and immune invasion. E2 from the J6 genotype has 11 glycosylation sites. Four of the glycosylation sites are in the flexible amino terminal region, which were deleted, and seven are in the core domain (N5-N11). The location of N7, N8, N10 and N11 are modeled in the final E2 core structure. All of these glycans are present in loop areas, indicating that these sites are solvent exposed and flexible. Mutagenesis studies in HCVcc have shown that N6, N8, and N10 are integral for virus infectivity. Removal of the N6 site results in improved CD81 binding, while N8 and N10 mutations destabilize the protein and cause defective particle production 19. Both sheets have one critical glycosylation site, N8 in sheet A and N10 in sheet B. All four of the observed glycosylation sites are on the periphery of the core and are located on a highly basic surface (Fig. 3). The opposite surface is predominantly hydrophobic and highly conserved when compared to the basic surface. Furthermore, the epitope for antibodies (AR1, AR3A and AR3C) that inhibit E1E2 binding to CD81 is located at the interface of the hydrophobic and basic surfaces, including the N7 glycosylation site (Extended Data Fig. 7a). Interestingly, N7 is only 7 residues away from N6, which has a critical role in CD81 binding. Epitopes for antibodies (i.e., AR5) that block E1E2 heterodimerization are also found on the hydrophobic surface, making it highly plausible that this surface is interacting with E1 in the context of the viral particle 4.


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)

Surface features of E2The surface of the E2 core domain colored for (a and d) electrostatic-1 potential: blue (basic), white (neutral), red (acidic) at ± 5 kTe-1 and (b and e) sequence identity (green) from the alignment in Extended Data Fig. 2. Ribbon diagram highlighting the location of the N-linked glycosylation sites are shown (c and f). The orientation of d, e, and f as well as a, b, and c are identical. The orientation in d, e, and f is rotated 180° about a horizontal axis from the view in a, b and c.
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Related In: Results  -  Collection

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

Figure 10: Surface features of E2The surface of the E2 core domain colored for (a and d) electrostatic-1 potential: blue (basic), white (neutral), red (acidic) at ± 5 kTe-1 and (b and e) sequence identity (green) from the alignment in Extended Data Fig. 2. Ribbon diagram highlighting the location of the N-linked glycosylation sites are shown (c and f). The orientation of d, e, and f as well as a, b, and c are identical. The orientation in d, e, and f is rotated 180° about a horizontal axis from the view in a, b and c.
Mentions: HCV E2 is modified by N-linked glycosylation, which is necessary for proper folding and immune invasion. E2 from the J6 genotype has 11 glycosylation sites. Four of the glycosylation sites are in the flexible amino terminal region, which were deleted, and seven are in the core domain (N5-N11). The location of N7, N8, N10 and N11 are modeled in the final E2 core structure. All of these glycans are present in loop areas, indicating that these sites are solvent exposed and flexible. Mutagenesis studies in HCVcc have shown that N6, N8, and N10 are integral for virus infectivity. Removal of the N6 site results in improved CD81 binding, while N8 and N10 mutations destabilize the protein and cause defective particle production 19. Both sheets have one critical glycosylation site, N8 in sheet A and N10 in sheet B. All four of the observed glycosylation sites are on the periphery of the core and are located on a highly basic surface (Fig. 3). The opposite surface is predominantly hydrophobic and highly conserved when compared to the basic surface. Furthermore, the epitope for antibodies (AR1, AR3A and AR3C) that inhibit E1E2 binding to CD81 is located at the interface of the hydrophobic and basic surfaces, including the N7 glycosylation site (Extended Data Fig. 7a). Interestingly, N7 is only 7 residues away from N6, which has a critical role in CD81 binding. Epitopes for antibodies (i.e., AR5) that block E1E2 heterodimerization are also found on the hydrophobic surface, making it highly plausible that this surface is interacting with E1 in the context of the viral particle 4.

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