A model system to examine the expression and antiviral activity of trans-acting ribozymes in mammalian cells has been developed and evaluated. Hairpin ribozymes were engineered to cleave a specific site, identified by a combinatorial activity-based selection method, within genomic and subgenomic RNA species of Sindbis virus. Transiently transfected cells expressed moderate levels of ribozyme (ϳ50,000 molecules/cell) with predominant nuclear localization and a short halflife (23 min). Stable cell lines expressed ribozymes at modest levels (ϳ2,000 molecules/cell). Ribozyme-mediated RNA cleavage activity was detected in cell extracts. Clonal cell lines were challenged with recombinant Sindbis virus, and viral replication was examined using plaque formation and green fluorescent protein assays. Significant inhibition of viral replication was observed in cells expressing the active antiviral ribozyme, and lower levels of inhibition in control cells expressing inactive or irrelevant ribozymes. These findings are consistent with a model in which inhibition of viral replication occurs via ribozyme cleavage of viral RNAs, suggesting that ribozymes may represent useful antiviral agents.Small trans-acting ribozymes such as the hammerhead and hairpin can function as highly selective endoribonucleases, the specificity of which can be manipulated in the laboratory (for review, see Ref. 1) (2-3). This property has led to an exploration of the potential utility of ribozymes for the targeted inactivation of cellular and viral RNAs. Indeed, there is an urgent need for the development of novel and effective measures to combat existing and emerging viral pathogens. Several groups (4 -7) have described attempts to achieve ribozyme inhibition of viral replication, but success has been limited. In some cases, there remains uncertainty as to whether viral inhibition results from ribozyme cleavage of the targeted RNA, because other mechanisms of inhibition, such as antisense effects and interference with the expression of cellular genes that support viral replication, have not always been ruled out.Our laboratory has recently addressed two major issues that may limit RNA inactivation by engineered ribozymes: (i) intracellular catalysis (8, 9) and (ii) target site accessibility (13,14). Results have shown that hairpin ribozymes can catalyze efficient cleavage of RNA substrates within the cytoplasm of mammalian cells both in cis (8) and in trans (9). These studies were undertaken systematically to monitor intracellular ribozymemediated RNA cleavage and were accompanied by controls that included inactive ribozyme, uncleavable substrates, and active ribozymes with irrelevant substrate specificity. We concluded that the intracellular selectivity and biochemical requirements of the hairpin ribozyme appear to parallel closely those that have been elucidated in the test tube. The same conclusion has recently been shown to be true for cleavage and ligation of the hairpin ribozyme in yeast (11,12).To examine the issue of target site specificity, ...
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