The goal of this project is to develop a novel class of effective antiviral agents based on the (-)s TRSV hairpin ribozyme. Ribozymes are catalytic RNA molecules; many catalyze site-specific RNA cleavage reactions. We will determine the mechanism for substrate recognition and cleavage of the hairpin ribozyme, and use this information to rationally alter the specificity of the ribozyme by engineering sequence changes within its substrate-binding domain.
The Specific Aims of this proposal are to (1) Determine the substrate selection rules for the hairpin ribozyme, (2) Manipulate the sequence specificity of the ribozyme to generate ribozymes specific for a variety of viral RNA sequences, (3) Construct and evaluate the activity of multivariant ribozymes, (4) Develop a bacteriophage system for analyzing and optimizing antiviral activity of ribozymes in vivo, and (5) Use the bacteriophage system to optimize the activity of ribozymes against foreign sequences. Ribozymes for this study are based on the self-cleaving minus strand of satellite tobacco ringspot virus ((-)sTRSV). In preliminary work, we have engineered a major change in the substrate specificity of the ribozyme, so that it selectively attacks HIV-1 pol sequences. Potential advantages of antiviral ribozymes are several: (i) Ribozymes can potentially inhibit both viral infection and the expression of viral genes in cells that are already infected, (ii) Ribozymes may be highly selective agents, (iii) Ribozymes act catalytically, so that a single molecule of ribozyme may potentially destroy many molecules of viral RNA, (iv) Ribozymes are not susceptible to pleiotropic drug resistance, and (v) ribozyme technology should be widely applicable, so that ribozymes may be rapidly developed to combat infections by newly emerging or newly recognized viruses. In addition, this work will produce a large amount of valuable information concerning the structure and function of ribozymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030534-02
Application #
3145549
Study Section
Biochemistry Study Section (BIO)
Project Start
1990-12-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Seyhan, Attila A; Vitiello, Danielle; Shields, Michele T et al. (2002) Ribozyme inhibition of alphavirus replication. J Biol Chem 277:25957-62
zu Putlitz, J; Yu, Q; Burke, J M et al. (1999) Combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis B virus. J Virol 73:5381-7
Murray, J B; Seyhan, A A; Walter, N G et al. (1998) The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol 5:587-95
Yu, Q; Pecchia, D B; Kingsley, S L et al. (1998) Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes. The most effective ribozymes are not predicted by substrate selection rules. J Biol Chem 273:23524-33
Berzal-Herranz, A; Burke, J M (1997) Ligation of RNA molecules by the hairpin ribozyme. Methods Mol Biol 74:349-55
Yu, Q; Burke, J M (1997) Design of hairpin ribozymes for in vitro and cellular applications. Methods Mol Biol 74:161-9
Sargueil, B; Burke, J M (1997) In vitro selection of hairpin ribozymes. Methods Mol Biol 74:289-300
Sargueil, B; Pecchia, D B; Burke, J M (1995) An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex. Biochemistry 34:7739-48
Butcher, S E; Heckman, J E; Burke, J M (1995) Reconstitution of hairpin ribozyme activity following separation of functional domains. J Biol Chem 270:29648-51