Hepatitis C viral proteins affect cell viability and membrane permeability.
To determine the effect of hepatitis C virus (HCV) proteins on cell growth, Huh-7 cells were transfected with a full-length HCV cDNA (pMO9.6-T7 Rz) clone and HCV proteins were expressed using a replication-defective adenovirus that encodes the gene for the T7 RNA polymerase. Expression of HCV proteins from this full-length clone resulted in reduction in viability of transfected cells as measured by trypan blue viability assay. For identification and separation of cells expressing hepatitis C virus proteins by fluorescence microscopy and flow cytometry, GFP was cloned in the HCV full-length clone. Cells transfected with the HCV-GFP chimera clone produced high levels of accurately processed structural and nonstructural proteins similar to those of the HCV full-length clone, which could be detected by Western blot analysis. Cells expressing all HCV proteins lost membrane permeability and underwent apoptotic cell death, indicated by the appearance of a sub-G0 peak in cell cycle analysis, DNA fragmentation in a TUNEL assay, and microscopic detection of nuclear condensation. Using double-channel flow analysis we confirmed that high-level expression of HCV proteins affected membrane permeability and cell survival. These results suggest that expression of all structural and nonstructural proteins from HCV cDNA in hepatic cells induces apoptotic cell death, which might be an important event in chronic hepatitis infection in humans.
Kalkeri
G
G
Department of Pathology and Laboratory Medicine, Tulane University Health Science Center, New Orleans, Louisiana 70112, USA.
Khalap
N
N
Akhter
S
S
Garry
R F
RF
Fermin
C D
CD
Dash
S
S
D017209:Apoptosis; D002463:Cell Membrane Permeability; D002470:Cell Survival; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006526:Hepatitis C; D006801:Humans; D008164:Luminescent Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*
D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins