HCV NS5A inhibits interferon-alpha signaling through suppression of STAT1 phosphorylation in hepatocyte-derived cell lines.
OBJECTIVE
HCV NS5A appears to play an important role in HCV resistance to IFN-alpha but the molecular mechanism is not fully elucidated. Most studies regarding the involvement of signal transducer and activator of transcription 1 (STAT1) in inhibition of IFN-alpha signaling by NS5A were performed in non-hepatic cell lines and their relevance may be debatable.


METHODS
We analyzed the effects of NS5A on IFN-alpha signaling through STAT1 phosphorylation in three hepatocyte-derived cell lines, Hep3B, J5 and Huh7. Interaction of NS5A and STAT1 was also investigated with co-immunoprecipitation and confocal microscopy.


RESULTS
IFN-alpha induces STAT1 activation in Hep3B cells in a dose- and time-dependent manner. Transient or stable NS5A expression inhibits STAT1 phosphorylation in a dose-dependent manner in hepatocyte-derived cell lines, whereas the levels of STAT1 phosphorylation remain unchanged in non-hepatocyte HeLa and COS7 cells despite increasing amounts of NS5A. The NS5A may interact with STAT1, specifically, the N-terminal 488 amino acids of STAT1. Furthermore, NS5A inhibits activation of interferon-stimulated gene factor 3 (ISGF3) and interferon-stimulated response element (ISRE)-driven gene expression, as demonstrated by electrophoretic mobility shift assay and luciferase assay, respectively.


CONCLUSIONS
NS5A may interact with STAT1 and inhibit IFN-alpha signaling through suppression of STAT1 phosphorylation specifically in hepatocyte-derived cells.
Lan
Keng-Hsin
KH
Division of Gastroenterology, Department of Medicine, National Yang-Ming University, Taipei, Taiwan. khlan@vghtpe.gov.tw
Lan
Keng-Li
KL
Lee
Wei-Ping
WP
Sheu
Meei-Ling
ML
Chen
Ming-Yuan
MY
Lee
Yung-Lu
YL
Yen
Sang-Hue
SH
Chang
Full-Young
FY
Lee
Shou-Dong
SD
D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D016174:Hepacivirus / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology / Q000821:virology*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016898:Interferon-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D016898:Interferon-alpha; D011505:Protein-Tyrosine Kinases; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase