Combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis B virus.
A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.
zu Putlitz
J
J
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, and Harvard Medical School, Boston, Massachusetts 02129, USA.
Yu
Q
Q
Burke
J M
JM
Wands
J R
JR
D000998:Antiviral Agents / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003470:Culture Media; D015723:Gene Library; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007424:Intracellular Fluid; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D000998:Antiviral Agents; D003470:Culture Media; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D016337:RNA, Catalytic; D012367:RNA, Viral