The objective of this proposal is to study the catalytic mechanisms of ribonuclease P ribozymes and their applications for inhibition of viral gene expression. The applicant has constructed a ribozyme engineered from the catalytic RNA subunit of RNase P from E. coli. This new ribozyme cleaves RNA substrates, including viral mRNAs, that base pair to its guide sequence. It order to increase the cleavage efficiency, the applicant will conduct experiments to elucidate the mechanism of catalysis and substrate recognition. He will also test whether viral replication can be abolished by targeting an essential viral gene, ICP4. Ribozyme recognition of target mRNA will be studied by both in vitro kinetic and physical mapping analyses. Efficient ribozymes will be generated by either site-directed mutagenesis or in vitro selection. The requirements for efficient ribozyme activity in tissue culture will be defined and the sequence specificity determined. Finally, whether the engineered ribozyme can inhibit HSV-1 viral replication by abolishing ICP4 expression will be investigated. The long term goal is to develop a system to study RNA catalysis and its use as a gene-targeting tool in basic research and clinical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM054815-04
Application #
6017097
Study Section
Experimental Virology Study Section (EVR)
Project Start
1996-06-01
Project End
2001-05-31
Budget Start
1999-06-01
Budget End
2000-05-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Public Health
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Trang, Phong; Liu, Fenyong (2004) RNase P ribozyme as an antiviral agent against human cytomegalovirus. Methods Mol Biol 252:437-50