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Journal of Virology, July 1999, p. 5381-5387, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus

Jasper zu Putlitz,1,dagger Qiao Yu,2,Dagger John M. Burke,2 and Jack R. Wands1,*

Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, and Harvard Medical School, Boston, Massachusetts 02129,1 and Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, The University of Vermont, Burlington, Vermont 054052

Received 1 December 1998/Accepted 13 April 1999

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 × 105 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.

* Corresponding author. Mailing address: The Liver Research Center, 55 Claverick St., 4th Floor, Providence, RI 02903. Phone: (401) 444-2795. Fax: (401) 444-2939. E-mail: Jack Wands MD{at}Brown.edu.

dagger Present address: Department of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany.

Dagger Present address: Genetic Therapy, Inc., Gaithersburg, MD 20878.

Journal of Virology, July 1999, p. 5381-5387, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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