Gene therapy protocols risk the potential of gene activation due to retroviral insertional activation. The ability to retarget or limit the sites of retroviral integration though cannot be successfully achieved in the absence of the basic understanding of retroviral integration. This proposal studies the integration of murine leukemia virus (MuLV), the most common retroviral vector used in gene therapy protocols. Integration is a critical step in the viral life cycle which results in the stable insertion of the viral genome throughout the host DNA. There is no mechanism of excision of an integrated provirus and integration can lead to the disruption and/or activation of cellular genes. The proposal utilizes biochemical and genetic approaches to define the interactions which orchestrate the assembly of protein-DNA integrative complex.
Three specific aims are proposed which examine individual components of the integration complex.
The first aim focuses on the viral Integrase (IN) protein, which catalyzes the transesterification process. These studies aim at defining the domains of the protein required to assemble a synaptic complex with the viral and target DNAs. The second specific aim defines the structural and/or sequence requirements of the viral substrate, the inverted terminal repeats at the ends of the LTRs. Novel polyamides which sequence specifically bind within the minor groove of the MuLV LTR termini have been developed. The effect of these compounds on the structure of the MuLV LTR will be examined. The third specific aim modifies the MuLV IN protein to include sequences found to direct integration to heterochromatin, considered to be transcriptionally inactive. This approach will adapt the mechanism identified for the yeast Ty5 system to the retroviral particles. The potential of the Ty5 tag to target retroviral integration will be examined. To date, there is no known structure of a retroviral integrase with either the target or viral DNA. These studies examine critical aspects of the protein-DNA recognition needed to identify a stable synaptic complex for structural and functional analysis. This knowledge of retroviral integration is necessary for further manipulation to target and/or inhibit this requisite step in the viral life cycle.
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