title: Folding of hepatitis C virus E1 glycoprotein in a cell-free system.
abstract: 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 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation 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 reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation 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 is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.
authors.affiliation: IRIS Research Center, Chiron, 53100 Siena, Italy. marcello_merola@chiron.it
authors.forename: M
authors.initials: M
authors.lastname: Merola
authors.forename: M
authors.initials: M
authors.lastname: Brazzoli
authors.forename: F
authors.initials: F
authors.lastname: Cocchiarella
authors.forename: J M
authors.initials: JM
authors.lastname: Heile
authors.forename: A
authors.initials: A
authors.lastname: Helenius
authors.forename: A J
authors.initials: AJ
authors.lastname: Weiner
authors.forename: M
authors.initials: M
authors.lastname: Houghton
authors.forename: S
authors.initials: S
authors.lastname: Abrignani
mesh_terms: D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000821:virology; D010084:Oxidation-Reduction; D017510:Protein Folding; D014759:Viral Envelope Proteins / Q000737:chemistry*
chemical_list: C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus