A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1͞IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a ''disabled'' ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications. Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe morbidity or mortality in neonates and immunocompromised individuals (1). Infection by this virus accounts for one of the most common opportunistic diseases in patients with AIDS, CMV retinitis. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and novel treatment strategies (2, 3).Antisense nucleic acid molecules, including conventional antisense oligonucleotides and antisense ribozymes, are promising gene-targeting agents for specific inhibition of gene expression (4-8). Antisense molecules have been used as anti-HCMV agents to inhibit the expression of HCMV-essential genes and abolish viral replication (4, 9-12). External guide sequences (EGSs; refs. 13 and 14) are antisense oligoribonucleotides that have been used in conjunction with either ribonuclease P (RNase P) or the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA; ref. 15) to diminish the expression of several genes both in E. coli (16,17) and in mammalian cells (14,(18)(19)(20)(21). The EGS-based technology takes advantage of RNase P or M1 RNA to cleave a targeted mRNA when the EGS hybridizes to the target RNA (refs. 13 and 14; Fig. 1A). The EGSs, when expressed separately from the enzyme, have been shown recently to be effective in inhibiting the gene expression of herpes simplex virus 1 (HSV-1) and influenza virus and, in addition, in abolishing the replication of influenza virus (20,21). To increase the targeting efficiency, the EGS can be covalently linked to M1 RNA (i.e., its 3Ј end) to generate a sequence-specific...

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G 224 G 225 3 AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k cat /K m ) than that derived from the wild type ribozyme. Our results suggest that the mutated A 224 A 225 are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (1-3). For example, both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, while a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (4 -9). Thus, ribozymes can be used as a tool in both basic and clinical research, such as in studies of tumorigenesis and antiviral gene therapy.RNase P is a ribonucleoprotein complex responsible for the 5Ј maturation of tRNAs (10, 11). It catalyzes a hydrolysis reaction to remove a 5Ј leader sequence from tRNA precursors (ptRNA) 1 and several other small RNAs . In Escherichia coli, RNase P consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (10, 11). In the presence of a high concentration of salt, such as 100 mM Mg 2ϩ , M1 RNA acts as a catalyst and cleaves ptRNAs in vitro in the absence of C5 protein (12). Extensive studies with both phylogenetic and biochemical analyses have established models for the secondary and threedimensional structures of RNase P catalytic RNAs (13-16). These models provide a framework to identify the putative active site and substrate binding site, and to study the mechanism of RNase P catalytic RNAs.Studies on substrate recognition by RNase P have revealed that a small model substrate can be cleaved efficiently by M1 ribozyme (Fig. 1A). This model substrate contains a structure equivalent to the acceptor stem, the T-stem, the 3Ј CCA sequence, and the 5Ј leader sequence of a ptRNA molecule. Accordingly, M1 catalytic RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence (external guide sequence) (Fig. 1A) (17). Moreover, a sequen...

External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNA in vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application.Antisense technology has been shown to be a promising gene-targeting approach for use in basic research and clinical therapeutic applications. The gene-targeting agents used can be a conventional antisense oligonucleotide, an antisense catalytic molecule (ribozyme or DNA enzyme), or an antisense molecule with an additional (guide) sequence that targets the mRNA for degradation by endogenous RNases such as RNase L and RNase P (1-6). Antisense molecules with guide sequences have several unique features as gene-targeting agents. Targeting with these molecules results in irreversible cleavage and the cleavage can be in a catalytic fashion. Moreover, this targeting approach uses the cellular endogenous RNases (e.g. RNase P) for degradation of the target mRNA and, therefore, assures the stability and efficiency of the targeting enzymes in the cellular environment.Ribonuclease P (RNase P) is a ribonucleoprotein complex found in all organisms examined. It is one of the highly active enzymes in cells and is responsible for the maturation of 5Ј termini of all tRNAs, which account for approximately 2% of total cellular RNA (7,8). This enzyme catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA (9). Human RNase P has at least nine polypeptides and a RNA subunit (H1 RNA) (7, 10). One of the unique features of RNase P is its ability to recognize the structures, rather than the sequences, of the substrates, which allows the enzyme to hydrolyze different natural substrates in vivo or in vitro. Accordingly, any complex of two RNA molecules that resembles a tRNA molecule can be recognized and cleaved by RNase P (Fig. 1, A and B) (11, 12). One of the RNA molecules is called the external guide sequence (EGS).1 In principle, an mRNA sequence can be targeted for RNase P cleavage by using EGSs to hybridize with the target RNA and d...

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