The mechanisms of hepatocyte death and the events that lead to a high rate of chronic liver disease in patients infected with hepatitis C virus (HCV) are not known. In order to study the effect of HCV expression on hepatocyte growth and viability we developed various cell culture models. HepG2 cells transfected with full-length HCV RNA, antisense RNA or mock were assayed for HCV proteins by immunohistochemistry. HCV transfected cells exhibited reduced viability compared to controls. Apoptosis in a subset of cells expressing HCV proteins was demonstrated by morphological, ultrastructural analysis, flow cytometry and TUNEL assays. To improve the expression of HCV proteins in transfected cells, we established HepG2 cells constitutively expressing HCV proteins and screened for HCV RNA by RT-PCR. Constitutive expression of HCV proteins was confirmed by immunohistochemistry and immunofluorescence assays. Decreased viability of stable cells corroborated our earlier results. Fragmented and condensed chromatin in the nucleus of these cells suggested apoptotic cell, death. An inducible system using a fluorescent marker for HCV expression was further developed in order to be able to identify and sort positive cells by flow cytometry. Coding sequence of green fluorescent protein (GFP) was PCR amplified and introduced into a full-length hepatitis C virus cDNA clone (pMO9.6-T7) in frame with the core region. The chimera was transfected to Huh7 cells and expressed using a replication defective adenovirus encoding T7 RNA polymerase. Production of RNA transcripts from the T7 promoter of the transfected HCV cDNA was confirmed by a ribonuclease protection assay. Cells transfected with this chimeric plasmid produced very high levels of accurately processed structural and nonstructural proteins, as revealed by Western blot analysis and were colocalized along with the core-green fluorescent fusion protein in the cytoplasm of transfected cells. High level expression of HCV proteins from this chimeric full-length clone could be correlated to decreased viability of transfected cells due to apoptosis. Apoptolic nuclei were observed by fluorescent, histochemical and ultrastructural studies. All these studies indicate that hepatic cells expressing HCV proteins undergo apoptotic cell death in vitro, and suggest that this form of cell death may contribute to pathogenesis of chronic hepatitis C virus infection in vivo.
展开▼
