Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.
Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.
Dubuisson
J
J
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
Hsu
H H
HH
Cheung
R C
RC
Greenberg
H B
HB
Russell
D G
DG
Rice
C M
CM
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D017931:DNA Primers; D005347:Fibroblasts; D015870:Gene Expression; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012845:Sindbis Virus / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D014759:Viral Envelope Proteins