siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.
RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.
Konishi
Masayoshi
M
Division of Gastroenterology & Hepatology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Wu
Catherine H
CH
Kaito
Masahiko
M
Hayashi
Kazuhiko
K
Watanabe
Shozo
S
Adachi
Yukihiko
Y
Wu
George Y
GY
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000821:virology; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017354:Point Mutation; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000235:genetics; D012093:Replicon / Q000235:genetics*; D012254:Ribavirin / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral; D012254:Ribavirin; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase