JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by zu Putlitz, J.
Right arrow Articles by Wands, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by zu Putlitz, J.
Right arrow Articles by Wands, J. R.

 Previous Article  |  Next Article 

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.



This article has been cited by other articles:




Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 1999 by the American Society for Microbiology. All rights reserved.