No abstract

The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coil has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by proteins extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by -80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA.[Key Words: RNA enzyme; gene inactivation; herpes simplex virus; thymidine kinase] Received November 3, 1994; revised version accepted January 20, 1995.Ribonuclease P (RNase P) is a ribonucleoprotein that is essential for the biosynthesis of the 5' termini of tRNAs (Altman et al. 1993). In Escherichia coli this enzyme consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (Dart et al. 1992;Altman et al. 1993). M1 RNA itself cleaves precursors to tRNAs (ptRNAs) and other small RNAs in E. coli (Fig. 1A) in vitro (Guerrier-Takada et al. 1983;Liu and Altman 1994). A minimal, model substrate for M1 RNA and the RNase P holoenzyme (M1 RNA plus C5 protein) contains only the equivalent of the acceptor stem and the T stem of a ptRNA molecule (McClain et al. 1987;Forster and Altman 1990a). Moreover, the 5' leader sequence and the 5' proximal sequence of the acceptor stem, when hydrogen-bonded as a separate oligonucleotide to the 3' proximal sequence of the acceptor stem [a construct designated an external guide sequence (EGS)], can also be cleaved by M1 RNA or RNase P (Fig. 1B). This observation led us to a novel strategy for gene inactivation whereby an EGS that binds to and targets a specific mRNA (e.g., [3-galactosidase mRNA) such that the mRNA can be cleaved by M1 RNA or RNase P (Li et al. 1992). One limitation of this method is the relatively weak binding of the target RNA to the enzyme, namely, M1 RNA. However, if the EGS is included as part of the enzyme (M1 RNA1, it becomes possible to increase both the strength of binding of the substrate and the efficiency of cleavage. Self-cleaving conjugates of M1 RNA and ptRNA have been described previously (Altman 1989; ~Corresponding author. Kikuchi et al. 1993;Frank et al. 1994) but have not, to our knowledge, been exploited as sequence-specific endonucleases in the manner described here.To further our understanding of catalysis by M1 RNA and also to design strategies for efficient cleavage by EGS-based RNase P, we constructed a derivative of M1 RNA, which we call M1GS RNA, by linking a guide sequence (GS) to M1 RNA (Fig. 1C). We show that M1GS RNA can act as a sequence-specific endonuclease and can cleave target RNAs that base-pair with the GS just as group I introns do (Zaug et al. 1986...

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...