FIG. 4.

Compartmentation of HCV structural proteins within DRM fractions. Lysates of HCVcc-infected cells were either treated with 1% TX-100, either on ice or at 37°C, or left untreated, followed by sucrose gradient centrifugation. (A and B) For each fraction, the amount of core protein was determined by an enzyme-linked immunosorbent assay (A), and E1, calnexin, and caveolin-2 were analyzed by Western blotting (B). The amount of core protein in each lysate (TX-100, 37°C; TX-100, 4°C; Untreated) was assigned the arbitrary value of 100%. M, membrane; NM, nonmembrane; DS, detergent soluble. (C) Lysates of 293T cells expressing HCV E1 or E2 protein were either treated with 1% TX-100, either on ice or at 37°C, or left untreated, followed by discontinuous sucrose gradient centrifugation. Each fraction was concentrated in a Centricon YM-30 filter unit and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against calnexin, caveolin-2, Myc (E1), or FLAG (E2). (D) (Top) Structures of HCV envelope genes used. Amino acid positions of HCV are indicated. Signal sequence, transmembrane (TM), and cytoplasmic tail (CT) domains of VSV G protein are shown. (Bottom) Cell lysates expressing chimeric HCV E1 or E2 protein were treated with 1% TX-100 on ice or left untreated, followed by discontinuous sucrose gradient centrifugation. It has been reported that VSV-G is not associated with lipid (39). Calnexin, caveolin-2, and chimeric glycoproteins (chimeric E1 and chimeric E2) were analyzed by immunoblotting. Fractions are numbered from 1 to 9 in order from top to bottom (light to heavy).