Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections encountered in patients with AIDS. Disseminated HCMV infection in these patients is usually associated with gastroenteritis, pneumonia, and sight-threatening retinitis. However, genetic analyses of this virus to characterize gene products essential for viral replication and pathogenesis and identify new targets for drug development have been hampered since it grows slowly and propagates only in human culture. Meanwhile, the emergence of drug-resistant HCMV strains to currently available drugs (e.g. ganciclovir and foscarnet) has posed a need to develop new drugs and novel strategies to combat HCMV infections. This proposal represents our continued effort to develop ribonuclease P (RNase P) ribozyme as a gene targeting tool for studies of the functions of HCMV genes and as a therapeutic agent for the treatment of HCMV infections. Recently, we have shown that RNase P ribozyme (M1GS RNA) can cleave the mRNAs coding for the HCMV transcription regulator IE1 and IE2, and block viral gene expression and growth. Further studies on the catalytic mechanism and sequence specificity of these ribozymes are necessary in order to improve their efficacy in inhibiting HCMV gene expression and replication. In this research program, we propose to develop a novel selection system to generate ribozyme variants that are highly active in cells, and to determine whether the generated ribozymes can be used as gene-targeting tools for complete inhibition of HCMV replication. In the initial study, ribozymes that effectively block CMV gene expression and abolish viral growth will be generated using our novel selection system. Then, the antiviral activity of these generated ribozymes will be determined and their mechanisms in blocking viral replication will be investigated. Furthermore, in vitro biochemical studies will be carded out to investigate how these generated ribozymes efficiently cleave their target mRNAs under physiological cellular conditions. Finally, the efficacy and sequence-specificity of these generated ribozymes in tissue culture will be determined. These studies will generate novel ribozyme variants that are highly active in cells and can be used for gene-targeting applications. Moreover, these studies will reveal the mechanism of how the ribozymes achieve high cleavage efficiency in cellular environment and facilitate the development of M1GS ribozymes as therapeutic agents for the inhibition of gene expression and replication of HCMV and other human viruses.
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