PMID: 11337040May 5, 2001Paper

Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone

Journal of Virological Methods
J MyungSrikanta Dash

Abstract

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.

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Citations

Oct 21, 2010·Virologica Sinica·Susan L Uprichard
Oct 9, 2002·Journal of Virology·Ana Maria Rivas-EstillaAntonis E Koromilas
May 25, 2005·Liver International : Official Journal of the International Association for the Study of the Liver·Srikanta DashSwan N Thung
Nov 9, 2005·Microscopy Research and Technique·Srikanta DashRobert Garry
Jan 11, 2007·Journal of Viral Hepatitis·P SheehyL J Fanning
May 6, 2004·Experimental and Molecular Pathology·Ramesh PrabhuSrikanta Dash
Nov 9, 2011·Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases·Huma TariqIshtiaq Qadri
Oct 7, 2004·Analytical Biochemistry·Mirella L Meyer-FiccaJan-Heiner Küpper
Apr 30, 2003·Antiviral Chemistry & Chemotherapy·Robert W KingClaudio Pasquinelli
Dec 6, 2001·Experimental and Molecular Pathology·G KalkeriS Dash

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