Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone.
An inducible in vitro cell culture system was developed to assay HCV replication by direct biochemical means. A transcription plasmid containing a T7 promoter at the 5' end, full-length cDNA of the HCV genome, a ribozyme sequence from the antigenomic strand of hepatitis delta virus and a T7 terminator was prepared. To facilitate high-level transcription of HCV RNA, HepG2 cells were infected with replication deficient adenovirus containing the T7 RNA polymerase gene and later transfected with the transcription plasmid containing the full-length HCV genome. This transfection-based cell culture system expressed high levels of HCV structural (core, El and E2) and non-structural proteins (NS3 and NS5B) detectable by Western blot and immunofluorescence assays. Production of HCV RNA transcripts and presence of replicative negative strand of HCV was confirmed by ribonuclease protection assay indicating replication of HCV in the transfected HepG2 cell. The transfected HepG2 cells assembled 50-60 nm virus-like particles, which could be aggregated by anti-E2 antibodies. This model can be utilized for studying mechanisms of HCV replication, assembly of HCV particles and to test potential anti-HCV compounds.
Myung
J
J
Department of Pathology and Laboratory Medicine, Health Sciences Center, Tulane University, 1430 Tulane Avenue, , New Orleans, LA 70112, USA.
Khalap
N
N
Kalkeri
G
G
Garry
R
R
Dash
S
S
D003001:Cloning, Molecular; D004279:DNA, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000032:analysis; D017361:Viral Nonstructural Proteins / Q000032:analysis; D014771:Virion; D019065:Virus Assembly; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D004279:DNA, Viral; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C087035:E1 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C091015:glycoprotein E2, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase