Expression of E1E2 on hepatitis C RNA-containing particles released from primary cultured human hepatocytes derived from infected cirrhotic livers.
OBJECTIVE
To determine whether liver-derived hepatitis C RNA-containing particles express the E1E2 discontinuous antigenic determinant defined by unique monoclonal antibody (mAb) D32.10 which recognizes three highly conserved segments in E1 (aa297-306) and E2 (aa480-494 and aa613-621) envelope glycoproteins.


METHODS
Human hepatocytes were isolated from HCV-infected cirrhotic explanted livers. The liver-derived hepatitis C virus (HCV) particles released from three distinct cultures (genotypes 1b and 2b) were characterized. HCV RNA+ was quantified by real-time RT-PCR. The E1E2 antigenic activity was assessed by indirect ELISA and immunoblotting using D32.10. The density distributions of HCV RNA and E1E2 antigen were determined by isopycnic sucrose density gradients. HCV E1E2, E2 and core antigens were detected in the cells by immunochemical staining.


RESULTS
Liver-derived HCV particles contained HCV RNA (10⁶-10⁷ copies/mg of protein) and core proteins and expressed the E1E2/D32.10 epitope. HCV RNA and E1E2 cosedimented between 1.15 and 1.25 g/ml in sucrose gradients. Moreover, the mAb D32.10 detected E1E2 by immunostaining in HCV-infected hepatocytes in parallel with E2 and core antigens.


CONCLUSIONS
Our results provide evidence that the mAb D32.10 recognizes E1E2 envelope complexes expressed in the cell cytoplasm and on the surface of HCV RNA-containing particles released from short-term cultures of in vivo infected hepatocytes.
Ndongo
Ndiémé
N
INSERM, U871, Lyon, France.
Selliah
Subjini
S
Berthillon
Pascale
P
Raymond
Valérie-Ann
VA
Trépo
Christian
C
Bilodeau
Marc
M
Petit
Marie-Anne
MA
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D002500:Centrifugation, Isopycnic; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006526:Hepatitis C / Q000235:genetics / Q000821:virology; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology; D006801:Humans; D008103:Liver Cirrhosis / Q000503:physiopathology; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012367:RNA, Viral; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; C116123:E1E2 peptide; D000939:Epitopes; D010455:Peptides; D012367:RNA, Viral; D014759:Viral Envelope Proteins