Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway.
BACKGROUND
The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated.


RESULTS
Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones.


CONCLUSIONS
The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.
Department of Pathology and Laboratory Medicine, Tulane University of Health Sciences Center, 1430 Tulane Ave, New Orleans, LA 70112, USA.
Sidhartha
S
Hazari
Partha K
PK
Chandra
Bret
B
Poat
Sibnarayan
S
Datta
Robert F
RF
Garry
Timothy P
TP
Foster
Gus
G
Kousoulas
Takaji
T
Wakita
Srikanta
S
Dash
D002460:Cell Line; D017930:Genes, Reporter; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000276:immunology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000276:immunology*; D053612:Janus Kinases / Q000172:deficiency / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050791:STAT Transcription Factors / Q000172:deficiency / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D049452:Green Fluorescent Proteins; D016898:Interferon-alpha; D053612:Janus Kinases; D011993:Recombinant Fusion Proteins; D050791:STAT Transcription Factors; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase