Recently, a cryo-EM structure of a?full-length membrane-extracted E1E2 of genotype 1a was reported in complex with a set of antibodies that included heterodimer-specific antibody AR4A7. biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development. Subject terms: Hepatitis C virus, Cryoelectron FLJ14936 Mitragynine microscopy, Viral proteins, Protein vaccines HCV vaccine development has been challenged by difficulties in the biochemical preparation of E1E2 ectodomains. Here, the authors structurally characterize an engineered soluble E1E2 ectodomain complexed with broadly neutralizing antibodies, revealing it adopts a native fold amenable for vaccine design. Introduction An estimated 58 million people are infected with hepatitis C virus (HCV)1. Approximately 75% of infections become chronic and in turn can lead to cirrhosis or hepatocellular carcinoma, a major cause of liver-related deaths. The development of direct-acting antivirals provides a possible cure for contamination but does not prevent reinfection. Additionally, direct-acting antiviral treatment is Mitragynine usually inaccessible for the majority of the infected population, with less than 10% of the global population having access to treatment due to healthcare and financial constraints. These limitations suggest that the most viable method for controlling HCV infections worldwide is usually through the development of a prophylactic vaccine2C4. Although HCV is usually classified as a flavivirus, its two surface envelope glycoproteins, E1 and E2, do not appear to share sequence or structural features with other flavivirus type II fusion glycoproteins5,6. Both E1 and E2 are single-pass transmembrane proteins with N-terminal ectodomains of 160 and 330 residues, respectively, and C-terminal transmembrane domains of roughly 30 residues. E1 and E2 form heterodimers on the surface of the virion and have been proposed to form higher-order trimers, although a recent structure of membrane-extracted E1E2 heterodimer did not reveal higher-order oligomers7C10. Both E1 and E2 subunits are heavily glycosylated, with E1 made up of 5 to 6 predicted N-linked glycosylation Mitragynine sites and E2 between 9 to 11, depending on the genotype. Numerous conserved cysteines within both subunits mediate extensive disulfide bonding networks and have been suggested to be essential for viral entry but not for heterodimerization11,12. The E2 subunit interacts with various cellular entry receptors, including tetraspanin CD81, scavenger receptor-B1, occludin, and claudin. The E1 subunit has been presumed to play a role in the fusion of the viral and host membranes during entry, although sequence homology to other membrane fusogens is usually difficult to discern13C15. Multiple studies have identified broadly neutralizing antibodies (bnAbs) against HCV both from acute and chronic contamination, with the former being associated with viral clearance16. Epitope mapping and structural characterization of such bnAbs have defined antigenic regions (AR) or antigenic domains that they target, which fall on the individual E1 or E2 subunits or depend on E1E2 heterodimers for recognition5C7,16C22. Varying nomenclatures have been used to describe these sites, including AR 1-5, antigenic domains A-E, and epitopes I-III. AR3 or antigenic domains B, D, and E on E2, collectively known as the E2 neutralizing face, are highly conserved and overlap with the CD81 host receptor binding region16,23. AR4 and AR5 outside the receptor binding region are also targets of bnAbs, but ones that depend on an intact E1E2 heterodimer for recognition. These include antibodies AR4A and AR5A, as well as antibodies HEPC111 and HEPC13016,18,22. While heterodimer-specific bnAbs do not prevent CD81 binding, they are among the most broadly neutralizing antibodies identified to date18. Structural characterization of antigenic targets on E2 has provided a great deal of information on sites of immune vulnerability around the virus, notably for bnAb-targeting epitopes overlapping the CD81 receptor binding domain name20,24C29. Development of an HCV vaccine has nonetheless confirmed challenging due to multiple factors2,16,30. These include a high degree of E1E2 genetic diversity (7 genotypes and over 90 subtypes), conformational plasticity, glycan shielding of neutralizing epitopes, presence of immunodominant non-neutralizing epitopes, and a proclivity for aggregation2,31C35..