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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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Functional analyses of eE2 and E2 core(b) Antibodies from patient sera infected with HCV genotype 2 show a concentration dependent binding to eE2 (red) whereas healthy donor sera exhibits 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 (SEM). (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 SEM. (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 SEM. (e) To rule out the possibility of toxic effects from the recombinant proteins, the cell viability was measured as described in Material and 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 amino-terminal region of eE2, blocks infection. The measurements were done in triplicate with the error bars representing the SEM.
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Figure 4: Functional analyses of eE2 and E2 core(b) Antibodies from patient sera infected with HCV genotype 2 show a concentration dependent binding to eE2 (red) whereas healthy donor sera exhibits 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 (SEM). (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 SEM. (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 SEM. (e) To rule out the possibility of toxic effects from the recombinant proteins, the cell viability was measured as described in Material and 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 amino-terminal region of eE2, blocks infection. The measurements were done in triplicate with the error bars representing the SEM.

Mentions: Solution-based studies using limited proteolysis and hydrogen deuterium exchange (HDX) demonstrated that approximately 80 amino acids on the amino terminus (384-463) from hypervariable region (HVR) 1 through HVR2 are exposed and flexible. This region includes conserved sequences implicated in binding to the cellular receptors (SR-BI and CD81) as well as several epitopes for neutralizing antibodies (Fig. 1 and Extended Data Figs. 2, 3) 7-11. Various amino-terminal deletions were produced to minimize regions of disorder while preserving an even number of cysteines, potentially allowing them to form intramolecular disulfide bonds. All constructs were screened for aggregation by non-reducing SDS-PAGE and SEC. E2 core (456-656) is soluble, monomeric, and maintains similar secondary structure content when compared with eE2 as determined by reactivity towards HCV infected patient sera (Extended Data Fig. 4a-b) and circular dichroism (data not shown). However, in contrast to eE2, CD81 binding affinity and the efficiency of inhibition of HCVcc entry was diminished for the E2 core (Extended Data Fig. 4c-e). This suggests that the amino-terminus of eE2 is critical for CD81 interaction and likely undergoes a transition from disorder to order upon binding. Alternatively, the amino-terminal region may also be ordered through interactions with other factors, e.g. E1, apolipoproteins, lipids, cellular receptors, or antibodies.


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)

Functional analyses of eE2 and E2 core(b) Antibodies from patient sera infected with HCV genotype 2 show a concentration dependent binding to eE2 (red) whereas healthy donor sera exhibits 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 (SEM). (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 SEM. (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 SEM. (e) To rule out the possibility of toxic effects from the recombinant proteins, the cell viability was measured as described in Material and 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 amino-terminal region of eE2, blocks infection. The measurements were done in triplicate with the error bars representing the SEM.
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Related In: Results  -  Collection

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

Figure 4: Functional analyses of eE2 and E2 core(b) Antibodies from patient sera infected with HCV genotype 2 show a concentration dependent binding to eE2 (red) whereas healthy donor sera exhibits 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 (SEM). (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 SEM. (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 SEM. (e) To rule out the possibility of toxic effects from the recombinant proteins, the cell viability was measured as described in Material and 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 amino-terminal region of eE2, blocks infection. The measurements were done in triplicate with the error bars representing the SEM.
Mentions: Solution-based studies using limited proteolysis and hydrogen deuterium exchange (HDX) demonstrated that approximately 80 amino acids on the amino terminus (384-463) from hypervariable region (HVR) 1 through HVR2 are exposed and flexible. This region includes conserved sequences implicated in binding to the cellular receptors (SR-BI and CD81) as well as several epitopes for neutralizing antibodies (Fig. 1 and Extended Data Figs. 2, 3) 7-11. Various amino-terminal deletions were produced to minimize regions of disorder while preserving an even number of cysteines, potentially allowing them to form intramolecular disulfide bonds. All constructs were screened for aggregation by non-reducing SDS-PAGE and SEC. E2 core (456-656) is soluble, monomeric, and maintains similar secondary structure content when compared with eE2 as determined by reactivity towards HCV infected patient sera (Extended Data Fig. 4a-b) and circular dichroism (data not shown). However, in contrast to eE2, CD81 binding affinity and the efficiency of inhibition of HCVcc entry was diminished for the E2 core (Extended Data Fig. 4c-e). This suggests that the amino-terminus of eE2 is critical for CD81 interaction and likely undergoes a transition from disorder to order upon binding. Alternatively, the amino-terminal region may also be ordered through interactions with other factors, e.g. E1, apolipoproteins, lipids, cellular receptors, or antibodies.

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