In the HCV envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acids (aa) 270 and 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the aa314-342 region, which has been shown to contain two alpha helices (α2 and α3) by NMR studies. Twenty out of the twenty-two generated mutants were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI co-receptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix 315-324 residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its co-receptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry. [ABSTRACT FROM AUTHOR]