Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.
RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.
Hamazaki
Hiroyuki
H
Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Ujino
Saneyuki
S
Miyano-Kurosaki
Naoko
N
Shimotohno
Kunitada
K
Takaku
Hiroshi
H
D001483:Base Sequence; D045744:Cell Line, Tumor; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004722:Endoribonucleases / Q000032:analysis; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000737:chemistry*; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon; D051196:Toll-Like Receptor 3 / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000378:metabolism
D034741:RNA, Small Interfering; D012367:RNA, Viral; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D014764:Viral Proteins; D019892:eIF-2 Kinase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease