Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis

PLoS Pathogens

Kelli Jackson¹  Radhika Gopal¹  Erick Giang¹  Andrew Honda¹  Netanel Tzarum²  Ian A. Wilson²  Leopold Kong²  Trevor Barnes³  Mansun Law³  Andrew Ettenger³  Jennifer M. Pfaff³  Johnathan Guest³  Edgar Davidson³  Benjamin J. Doranz⁴ 

[1] Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States of America;Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States of America;Integral Molecular, Inc., Philadelphia, PA, United States of America;The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, United States of America

关键词: Flow cytometry;    Enzyme-linked immunoassays;    Alanine;    Crystal structure;    Antibodies;    Binding analysis;    Mutagenesis;    Point mutation;   

DOI  :  10.1371/journal.ppat.1006735

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.

【 授权许可】

CC BY