Activation of interferon-stimulated response element in huh-7 cells replicating hepatitis C virus subgenomic RNA.
Interferon-alpha (IFN(alpha)) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFN(alpha) was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFN(alpha). These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.
Pai
Mirabel
M
Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
Prabhu
Ramesh
R
Panebra
Alfredo
A
Nangle
Sarah
S
Haque
Salima
S
Bastian
Frank
F
Garry
Robert
R
Agrawal
Krishna
K
Goodbourn
Steve
S
Dash
Srikanta
S
D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020218:Response Elements; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation; D014779:Virus Replication
D016898:Interferon-alpha; D049409:Luciferases, Firefly