The C-terminal amphipathic α-helix in E^rns is important to compensation for the role of apolipoproteins in the infectious particle formation of HCV.

(A) The cDNA constructs for expression of HA-tagged E^rns (HA-E^rns) mutants; the full length of E^rns with the signal sequence of the C-terminal 30 amino acids of core protein (E^rns), and without the signal sequence (Δsig), the ectodomain of E^rns with the signal sequence (Ecto), and the C-terminal amphipathic α-helix of E^rns (Hel). (B) Expression of ApoE and the HA-E^rns mutants was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (C) and extracellular infectious titers (D) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. (E) The insertion of two proline residues (204P/210P) in the Hel mutant containing the C-terminal amphipathic α-helix of HA-E^rns of CSFV was generated to examine the significance of the membrane-binding ability in the formation of HCV particles. Expression of Hel, Hel (204P/210P), and ApoE was determined by immunoblotting at 48-h post-transduction of lentiviruses into the BE-KO cells. Intracellular HCV RNA (F) and extracellular infectious titers (G) were determined at 72-h post-infection with JFH1 HCV at an MOI of 1 by qRT-PCR and focus-forming assay, respectively. In all cases, asterisks indicate significant differences (* p < 0.01) versus the results of the control cells.