Folding of hepatitis C virus E1 glycoprotein in a cell-free system

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 a nd its folding, whereas knowledge of E1 folding remains fragmentary. We use d a cell-free in vitro translation system to study E1 folding and asked whe ther the flanking proteins, Core and E2, influence this process. We transla ted the polyprotein precursor, in which the Core is N-terminal to E1, and E 2 is C-terminal, and found that when the core protein was present, oxidatio n of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous r eports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially fo lded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lo wer efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 coul d be detected. Release from calnexin and calreticulin was used to assess fo rmation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that i s affected not only by E2, as previously shown, but also by the Core. The f olding of viral proteins can thus depend on complex interactions between ne ighboring proteins within the polyprotein precursor.