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J. Biol. Chem., Vol. 278, Issue 39, 37265-37274, September 26, 2003

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Engineered RNase P Ribozymes Are Efficient in Cleaving a Human Cytomegalovirus mRNA in Vitro and Are Effective in Inhibiting Viral Gene Expression and Growth in Human Cells^*

Hua Zou, Jarone Lee, Sean Umamoto, Ahmed F. Kilani, Joseph Kim, Phong Trang , Tianhong Zhou and Fenyong Liu 

From the Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, University of California, Berkeley, California 94720

By using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a ribozyme variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) major transcription regulatory proteins IE1 and IE2. The variant is about 90 times more efficient in cleaving the IE1/IE2 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Our results provide the first direct evidence that a point mutation at nucleotide position 80 of RNase P catalytic RNA from Escherichia coli (U⁸⁰ C⁸⁰) increases the rate of chemical cleavage, and another mutation at nucleotide position 188 (C¹⁸⁸ U¹⁸⁸) enhances substrate binding of the ribozyme. Moreover, the variant is more effective in inhibiting viral IE1 and IE2 expression and growth in HCMV-infected cells than the wild type ribozyme. A reduction of about 99% in the expression level of IE1 and IE2 and a reduction of 10,000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of less than 10% in IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered RNase P ribozyme variants are highly effective in inhibiting HCMV gene expression and growth. These results also demonstrate the feasibility of engineering highly effective RNase P ribozymes for gene targeting applications, including anti-HCMV gene therapy.

Received for publication, April 4, 2003 , and in revised form, July 15, 2003.

^* This work was supported in part by grants from the March of Dimes Birth Defects Foundation, grants from the American Heart Association, and Grants AI41927 and GM54815 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a predoctoral fellowship from American Heart Association (Western States Affiliate).

Pew Scholar in Biomedical Sciences and a Scholar of Leukemia and Lymphoma Society. To whom correspondence should be addressed: School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720. Tel.: 510-643-2436; Fax: 510-643-9955; E-mail: liu_fy{at}uclink4.berkeley.edu.

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