Abstract
RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.
Highlights
Human cytomegalovirus (HCMV) is a common herpesvirus and usually affects individuals with compromised immune system [1]
We investigated the activity of the constructed ribozyme in generating cleavage of immediate-early protein 2 (IE2) mRNA sequence in vitro and in shutting down human cytomegalovirus (HCMV) infection in cells
Since most mRNA species inside cells form complex secondary structures and are associated with proteins, a targeting region must be accessible for ribozyme binding and catalytic cleavage of the target mRNA
Summary
Human cytomegalovirus (HCMV) is a common herpesvirus and usually affects individuals with compromised immune system [1]. This virus causes birth defects including mental problems [2]. New therapeutic approaches are needed to combat and control this important opportunistic pathogen. Gene targeting approaches using nucleic acid-based molecules to target specific mRNA sequences of choice represent promising therapeutic strategies [5,6]. One example is small interfering RNAs (siRNAs), which were used to target several human viruses effectively [5,7,8]. Ribozymes can inactivate viral mRNA sequences and diminish viral infection in human cells [9,10,11,12]
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