This proposal describes a research program to develop ribonuclease P (RNAseP) as a gene targeting tool for studies of the functions of HCMV genes and as a therapeutic agent for the treatment of HCMV infections. HCMV is one of the most common opportunistic infectious agents in AIDS patients. Genetic analyses of the virus have been hampered by its slow growth rate in cell culture. Although drugs for treatment of HCMV infection are available, resistance to the commonly used drugs ganciclovir and foscarnet is a problem and poses the challenge of developing new drugs and methods for treatment of HCMV infection. This proposal represents a research program to develop ribonuclease P as a gene targeting tool for studies of the functions of HCMV genes and as a therapeutic agent for treatment to HCMV infections. Recently, the P.I. has shown that the RNAse P RNA (M1GS RNA) cleaves RNA substrates including a herpes simplex virus mRNA that base pairs to its guide sequence. In this proposal efficient and sequence-specific M1GS RNAs will be generated to target the mRNAs encoding the protease which cleaves the capsid assembly protein during HCMV capsid formation, and the UL97 protein which phosphorylates ganciclovir and plays a central role in metabolizing this drug and controlling its effectiveness in cells. Targeting cleavage of these two mRNAs by M1GS ribozymes will carried out to study the role of these two proteins in the HCMV lytic cycle and determine whether they are essential for viral growth. These studies are also intended to reveal the mechanism of how the ribozymes achieve high cleavage efficiency and sequence-specificity, and explore the possibility of using M1GS RNA as a therapeutic agent for the inhibition of HCMV replication by abolishing the expression of viral essential genes.
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