Blocking Hepatitis C Virus Infection with Recombinant Form of Envelope Protein 2 Ectodomain

Abstract

More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains

“…In this regard, it has been described that the folding of the E2 ectodomain is independent of E1, but truncated E2 is required for the proper folding of E1 ectodomain ( Patel et al, 2001;Michalak et al, 1997). Hence, isolated properly folded E2 ectodomain produced either using the baculovirus-insect cell system (Rodriguez-Rodriguez et al, 2009) or using human HEK293 cells (Whidby et al, 2009) has been characterized and, very recently, the detailed structure of this truncated form bound to an antigen binding fragment has been described (Khan et al, 2014;Kong et al, 2013). Conversely, to date, no structural data of an isolated, properly folded, E1 ectodomain appears in the bibliography.…”

“…Despite that E1 and E2 have been expressed in several prokaryotic (Hüssy et al, 1997) or eukaryotic (Lorent et al, 2008;Mustilli et al, 1999;Michalak et al, 1997;Hüssy et al, 1996) cell lines, few data concerning to the structure of isolated proteins have been obtained. Most of the structural studies have been focused on the E2 protein, since, contrary to the full-length polypeptide, the E2 ectodomain has been characterized as an independent folding unit (Rodriguez-Rodriguez et al, 2009;Whidby et al, 2009). Thus, very recently, two different groups have described the crystal structure of the E2 core ectodomain in complex with antibodies (Khan et al, 2014;Kong et al, 2013).…”

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

“…These observations would indicate that HCV envelope glycoproteins cooperate to form a functional complex. However, E2 protein acquires a three dimensional structure compatible with a native conformation when it is expressed as an isolated form [6,9] which can block HCV infection in vitro [10]. Moreover, recent studies 5 carried out with E2 ectodomain (residues 384-661 of the polyprotein) produced, either in insect [11] or in mammalian cells [10], have shown that E2 can act as an independent folding unit.…”

“…However, E2 protein acquires a three dimensional structure compatible with a native conformation when it is expressed as an isolated form [6,9] which can block HCV infection in vitro [10]. Moreover, recent studies 5 carried out with E2 ectodomain (residues 384-661 of the polyprotein) produced, either in insect [11] or in mammalian cells [10], have shown that E2 can act as an independent folding unit. Structural characterization of the E2 ectodomain by means of circular dichroism [10][11][12] and infrared spectroscopy [12], indicates that E2 is mainly constituted by β-sheet and non-ordered structures.…”

“…Moreover, recent studies 5 carried out with E2 ectodomain (residues 384-661 of the polyprotein) produced, either in insect [11] or in mammalian cells [10], have shown that E2 can act as an independent folding unit. Structural characterization of the E2 ectodomain by means of circular dichroism [10][11][12] and infrared spectroscopy [12], indicates that E2 is mainly constituted by β-sheet and non-ordered structures. Based on gene organization HCV E2 envelope glycoprotein has been classified as a class II fusion protein [13].…”