FIG. 1.

Cell surface expression of E1 and E2. Unmodified HCV envelope glycoproteins were transiently expressed in HeLa cells. Briefly, cells were seeded overnight on glass coverslips and were infected with 5 PFU of recombinant vaccinia virus vTF7.3 per cell for 1 h at 37°C, followed by lipofection (Invitrogen) with the E1-E2 construct. Protein expression was analyzed 24 h postinfection (a to d). Alternatively, cells were only lipofected with the E1-E2 construct and protein expression was analyzed 24 h postlipofection (e to h). Cells were either fixed in 3% formaldehyde for 20 min at room temperature or fixed and permeabilized with methanol for 20 min at −20°C, followed by washing with 2% gelatin in phosphate-buffered saline. Cells were incubated with anti-E1 MAb A4 (1:100) (a, c, e, and g) or anti-E2 MAb H53 (1:100) (b, d, f, and h), followed by washing and incubation with a phycoerythrin (PE)-labeled goat anti-mouse immunoglobulin G secondary antibody (1:100) (Pierce). Coverslips were mounted on slides with Mowiol and were observed under a fluorescence microscope.