Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.Hepatitis C virus (HCV) is the sole member of the Hepacivirus genus within the family Flaviviridae. It is a major cause of community-acquired and posttransfusion hepatitis. More than 170 million people worldwide are seropositive for HCV, and only 20% of those infected are able to clear the virus. In the remaining 80% of individuals, the virus persists and can

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope. HCV, a member of the Flaviviridae family, has a 9-kb genome encoding a polyprotein precursor that is cleaved to yield three structural proteins, core, E1, and E2, together with at least six non-structural proteins. E1 and E2 are highly glycosylated membrane-anchored proteins that mediate viral entry. 2-6 E2 has been shown to bind to a number of cell surface molecules. 7-10 Whereas the exact mechanism of viral entry is unknown, mounting evidence indicates that CD81 and SR-BI are key molecules. 2,[4][5][6][11][12][13][14] In an infected individual, HCV exists as a viral quasispecies. 15 HCV can be classified into at least six major genotypes that exhibit extensive genetic variability, particularly in E1 and E2. 16 E1 and E2 are the principle targets for neutralizing antibodies, and identification of protective epitopes conserved across different strains of HCV is a major challenge for vaccine design. 17 A number of antibodies that are capable of blocking E2 binding to cells or cell receptors have been described, 18-23 some of which neutralize HCV entry in animal or in vitro models. 6,[24][25][26] The first hypervariable region of E2 contains potent neutralizing epitopes, and antibodies raised against

No abstract