Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus.
A recombinant baculovirus, RecBV-E, encoding the hepatitis C virus (HCV) envelope proteins, E1 and E2, controlled by the cytomegalovirus promoter was constructed. RecBVs can infect mammalian cells, but fail to express proteins or replicate because the viral DNA promoters are not recognised. The RecBV-E transduced 86% of Huh7 cells and 22% of primary marmoset hepatocytes compared with 35% and 0.4%, respectively, after DNA transfection. Several stable cell lines were generated that constitutively expressed E1/E2 in every cell. No evidence of E1/E2-related apoptosis was noted, and the doubling times of cells were similar to that of the parental cells. A proportion of the E1/E2 was expressed on the surface of the stable cells as determined by flow cytometry and was detected by a conformation-dependent monoclonal antibody. It is likely that the continued expression of E1/E2 in the stable cells resulted from integration of the RecBV DNA. Infection of Huh7 cells, in the absence of G418 selection, failed to result in expression of the foreign gene (in this case, eGFP) beyond 14-18 days. RecBVs that express HCV genes from a CMV promoter represent an effective means by which to transduce primary hepatocytes for expression and replication studies.
Martyn
J C
JC
Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia. martyn@burnet.edu.au
Dong
X
X
Holmes-Brown
S
S
Pribul
P
P
Li
S
S
Drummer
H E
HE
Gowans
E J
EJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D002144:Callithrix; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology; D011401:Promoter Regions, Genetic; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus