MECHANISM OF RETROVIRAL INTEGRATION

The aim of this project is to characterize the mechanism of integration of the retro virus Moloney-Murine Leukemia Virus (M- MuLV). Integration leads to the stable and efficient introduction of a DNA copy of the viral genome into the target DNA. This is a key determinant for the use of retro viral vectors as vehicles for gene therapy as well as a contributing factor toward the mutagenic and leukemogenic potential of the virus by disruption and/or activation of cellular genes. Through the understanding of the integration mechanism, the ability to limit the mutagenic potential of these viruses and vectors may be obtained. Integration requires the viral encoded Integrase (IN) protein as well as DNA sequences at the termini of the linear virus. Correct integration requires the coordination of the two viral ends and the target DNA thought the formation of multimeric IN complexes. Experiments are aimed at defining the domains of the M-MuLV IN protein responsible for the assembly of the oligomeric complexes. Of particular interest is the role of the HHCC finger region using complementation assays as well as the protein-protein and protein-DNA interactions of this domain. The assembly integration using a complementation assay is developed which would remove the potential for non-specific viral integrations. Biochemical characterization of the HHCC region with respect to dimerization and metal binding will assist in the purification of this domain for x-ray crystallographic studies. Upon infection, the reintegrative complex is found in the nucleus after passage through M phase. The composition of the reintegrative complex will be analyzed. The subcellular localization of the IN protein in the absence and presence of the viral proteins will be determined. Through the insertion of nuclear localization sequences into targeted viral proteins, the ability of the murine based retro viruses to infect non-replicating cells will be examined.