Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3.
BACKGROUND
Interferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system.


METHODS
An HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE).


RESULTS
The baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% +/- 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels.


CONCLUSIONS
IRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.
Yamashiro
Tsuyoshi
T
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan.
Sakamoto
Naoya
N
Kurosaki
Masayuki
M
Kanazawa
Nobuhiko
N
Tanabe
Yoko
Y
Nakagawa
Mina
M
Chen
Cheng-Hsin
CH
Itsui
Yasuhiro
Y
Koyama
Tomoyuki
T
Takeda
Yoshie
Y
Maekawa
Shinya
S
Enomoto
Nobuyuki
N
Sakugawa
Hiroshi
H
Watanabe
Mamoru
M
D015153:Blotting, Western; D045744:Cell Line, Tumor; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D050838:Interferon Regulatory Factor-3 / Q000502:physiology*; D010957:Plasmids; D034741:RNA, Small Interfering; D012093:Replicon; D014162:Transfection; D014779:Virus Replication / Q000502:physiology*
D050838:Interferon Regulatory Factor-3; D034741:RNA, Small Interfering