Characterization of truncated forms of hepatitis C virus glycoproteins.
Hepatitis C virus (HCV) glycoproteins (E1 and E2) both contain a carboxy-terminal hydrophobic region, which presumably serves as a membrane anchor. When they are expressed in animal cell cultures, these glycoproteins, in both mature complexes and misfolded aggregates, are retained in the endoplasmic reticulum. The effect of carboxy-terminal deletions on HCV glycoprotein secretion and folding was examined in this study. Sindbis and/or vaccinia virus recombinants expressing truncated forms of these glycoproteins ending at amino acids 311, 330, 354 and 360 (truncated E1), and 661, 688, 704 and 715 (truncated E2) were constructed. When expressed using Sindbis virus vectors, only truncated forms of E1 and E2 ending at amino acids 311 (E1t311) and 661 (E2t661), respectively, were efficiently secreted. Analysis of secretion of truncated forms of E2 glycoprotein expressed by vaccinia viruses indicated that significant secretion was still observed for a protein as large as E2t715. However, only secreted E2t661 appeared to be properly folded. Secreted HCV glycoprotein complexes were also detected in the supernatant of cell culture when E1t311 and E2t661 were coexpressed. Nevertheless, these secreted complexes, as well as E1t311 expressed alone, were misfolded. The effect of coexpression of E1 and E2 glycoproteins on each other's folding was evaluated with the help of a conformation-sensitive monoclonal antibody (for E2) or by analysing intramolecular disulfide bond formation (for E1). Our data indicate that the folding of E2 is independent of E1, but that E2 is required for the proper folding of E1.
Michalak
J P
JP
CNRS-UMR319, Institut de Biologie de Lille, France.
Wychowski
C
C
Choukhi
A
A
Meunier
J C
JC
Ung
S
S
Rice
C M
CM
Dubuisson
J
J
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D012845:Sindbis Virus / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C087035:E1 protein, Hepatitis C virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus