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In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface.

ABSTRACT: Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62-66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCV glycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.

SUBMITTER: Davis C

PROVIDER: S-EPMC3549482 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface.

Davis Christopher C Harris Helen J HJ Hu Ke K Drummer Heidi E HE McKeating Jane A JA Mullins Jonathan G L JG Balfe Peter P

Cellular microbiology 20120925 12

Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Re ...[more]

PMID: 22897233

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Project description:Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W(437)LAGLF(442), which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W(437)LAGLF(442) helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV.Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV.

Project description:The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

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In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface.

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In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface.

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