DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication.
OBJECTIVE
To investigate whether DNA-dependent activator of interferon-regulatory factors (DAI) inhibits hepatitis B virus (HBV) replication and what the mechanism is.


METHODS
After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plasmid, viral protein (HBV surface antigen and HBV e antigen) secretion was detected by enzyme-linked immunosorbent assay, and HBV RNA was analyzed by real-time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 (IRF3) phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-κB (NF-κB) activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Transwell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines.


RESULTS
Viral protein secretion was significantly reduced by 57% (P < 0.05), and the level of total HBV RNA was reduced by 67% (P < 0.05). The viral core particle-associated DNA was also dramatically down-regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-κB signaling was essential for DAI to elicit antiviral response in Huh7 cells. When the NF-κB signaling pathway was blocked by a NF-κB signaling suppressor (IκBα-SR), the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was independent of IRF3 signaling and secreted cytokines.


CONCLUSIONS
This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is associated with activation of NF-κB but independent of IRF3 and secreted cytokines.
Key Laboratory of Medical Molecular Virology, Ministry of Education and Ministry of Health, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Qi-Ying
QY
Chen
Ying-Hui
YH
Liu
Jian-Hua
JH
Li
Ze-Kun
ZK
Wang
Jiang-Xia
JX
Liu
Zheng-Hong
ZH
Yuan
D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006515:Hepatitis B virus / Q000235:genetics / Q000254:growth & development* / Q000276:immunology; D022781:Hepatocytes / Q000276:immunology / Q000378:metabolism* / Q000821:virology*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
D016207:Cytokines; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012367:RNA, Viral; D000072000:NF-KappaB Inhibitor alpha