Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide 1. HCV infection often results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. No vaccine is available and current therapies are effective against certain, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies 2-4. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an Fab at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of beta strands and random coil with two small alpha helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulfide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins while sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements upon exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

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.

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Hepatitis C virus (HCV) is the prototype member of the Hepacivirus genus within the family Flaviviridae. The virus is transmitted almost exclusively by the parenteral route, and acute infections, which are frequently subclinical, progress to chronicity in about 70% of cases. Persistent HCV carriers may develop liver cirrhosis, hepatocellular carcinoma, and end-stage liver disease. Despite an efficient preventive campaign based on the identification of HCV-infected blood donors, the prevalence of the virus among American young adults has not declined (35). Recent advances have improved treatment options for infections with certain viral genotypes, although limitations in efficacy remain and a preventive vaccine is not available.HCV is an enveloped virus, with a positive-sense, singlestranded RNA genome. The genome is translated into a polyprotein which is proteolytically processed into 10 individual proteins (reviewed in reference 64). The structural proteins, core protein (C) and two envelope glycoproteins (E1 and E2), form the physical viral particle; C functions to encapsidate the RNA genome, while E1 and E2 mediate virus attachment and entry into host cells. E1 and E2 are highly glycosylated type I transmembrane proteins with an N-terminal ectodomain. Residues within the transmembrane domains are important for heterodimerization and dimer retention in the endoplasmic reticulum (ER) (22,46). Replacement of the E1 or E2 transmembrane domains can direct transport of the corresponding chimeric proteins to the plasma membrane (1, 10). E2 also encompasses well-conserved antibody neutralization determinants, which are located near the binding sites for viral entry factors CD81 and scavenger receptor B1 (20,29). There is also some evidence for the existence of neutralizing determinants in E1 (45). Patient neutralizing antibodies have been identified that target virus interactions with its coreceptors and block glycoprotein-mediated membrane fusion (31).A major hurdle in the development of an effective HCV vaccine is the lack of an immunization strategy to elicit broadly protective antibodies and sustained cell-mediated immunity (32, 66). Studies with chimpanzees have shown the importance of total anti-E1/E2 antibody titers in conferring protection (15). Moreover, neutralizing immunity and functional CD4 ϩ and CD8 ϩ Tcell responses induced early in HCV infection correlate with clearance or viral control in patients (7,37,51).In contrast, the measles virus (MV) vaccine has an outstanding record of efficacy and safety. The MV Moraten vaccine strain is credited with the temporary elimination of indigenous measles transmission on the American continent (14), and the World Health Organization is implementing a global measles eradication program (72,73). After completion of the two-dose vaccination schedule, nearly 100% of recipients develop lasting neutralizing immunity that may be lifelong (2). In addition, the availability of established production methods makes MV an appealing platform for delivering foreign antigens (r...

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. T he specificity of retroviral vectors is initially conferred by the envelope (Env) protein present on the surface of virus particles. This Env protein is responsible for binding to a host cell receptor, thereby initiating virus entry. In gammaretroviruses, the Env protein is composed of a transmembrane protein (TM) and a surface protein (SU). For murine leukemia virus (MLV) and feline leukemia virus (FeLV) SUs, primary receptor binding is localized to the N-terminal half of SU (1, 2, 12). Critical residues for specificity are encoded within the N-terminal variable regions, VRA and VRB. Secondary receptor binding sites that promote viral infection have also been identified within the SU C terminus (3-5).FeLV Envs are classified as A, B, and C, originally based on interference assays (6, 7). The receptors for these three major subgroups have been identified as THTR1 (8), PiT1 (9), and FLVCR1 (10, 11), respectively, molecularly confirming the distinct receptor usage for each subgroup. For FeLV-A, 19 residues in the VRA can be replaced with 16 residues of FeLV-C VRA to sufficiently alter the binding specificity, thus switching receptor usage (12). In FeLV-A, the VRA and VRB each include a pair of cysteines, which have the potential to form intramolecular disulfide bonds (5). The cysteine contents of MLV and FeLV-B SUs are more complex; ecotropic MLV ...