Abstract
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.
Highlights
Hepatitis C virus (HCV) is a major global health concern with over 170 million people currently infected and an additional 3 million being infected each year
High-throughput flow cytometry is a well-established system that has been used in the study of many viral antigens, and was applied to probe the E1E2 mutant library with a panel of monoclonal antibodies (mAbs) and CD81-LEL [26]
The expression of the mutants was monitored by comparing mAb binding to wild-type E1E2 via the C-terminal V5 epitope fusion tag or mAbs to E1 and E2 continuous epitopes (A4, HCV1 and AP33)
Summary
Hepatitis C virus (HCV) is a major global health concern with over 170 million people currently infected and an additional 3 million being infected each year (reviewed in [1, 2]). While approximately 30% of infected individuals are capable of spontaneously clearing the virus, usually within the first 12 months of infection, the remainder generally develops life-long infection. Of those who progress to chronic infection, about 20% develop liver cirrhosis and 1–3% hepatocellular carcinoma, one of the leading causes of cancer mortality [2, 3]. The high rate of infection in endemic countries and the morbidity caused by subsequent liver damage[5], as well as underdiagnosis, costly treatments and high rate of reinfection [6, 7], highlight the need for an effective HCV vaccine to limit virus infection and spread
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