Human cytomegalovirus (HCMV), a member of the human herpesviruses, 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 to 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 the available drugs has posed a need to develop new drugs and novel strategies to combat HCMV infections. This proposal represents our research program 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 gene-targeting RNase P ribozyme (MIGS RNA) efficiently cleaves mRNA substrates in vitro, including the mRNAs coding for the HCMV major transcription regulator proteins, IE1 and lE2, and are effective in inhibiting the expression of these mRNAs and HCMV replication in cell culture. 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, highly efficient and sequence-specific MIGS RNAs will be generated to target the mRNAs encoding IE1/1E2 proteins and the protease (PR) which is essential for HCMV capsid maturation. Moreover, biochemical studies will be carried out to understand how these ribozymes achieve efficient cleavage activity and high sequence specificity. Finally, the efficacy and sequence-specificity of these ribozymes in inhibiting the expression of the viral mRNAs will be determined, and whether they are highly effective and specific in abolishing HCMV replication will be studied. This study will reveal whether MIGS ribozymes can be used as gene targeting tools to study the functions of HCMV essential genes and furthermore, will facilitate the development of these ribozymes as therapeutic agents for the inhibition of gene expression and replication of HCMV and other human viruses.
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