Murine-based retroviruses continue to be developed as viral vectors, despite their association with oncogenic transformation and insertional mutagenesis. This is highlighted in the past month with the association of xenotropic murine leukemia virus-related virus (XMRV) with human diseases including chronic fatigue syndrome and prostate cancer. Although comparative retroviral/lentiviral analysis of integration sites has noted differences in the positioning within the host chromosome, the mechanism for tethering the murine-based viruses to the host chromosomes is not understood. Structural analysis of proteins can provide essential insights into function. This proposal develops and extends preliminary structural analysis of the N-terminal domain (NTD) of the Moloney Murine Leukemia Virus Integrase (M-MuLV IN) protein to define the function of this domain within the preintegrative complex. The structural analysis of this domain will be extended to XMRV, which maintains structural homology with MuLV IN. The first focus of the research is structural studies of the IN NTD. The MuLV NTD is distinct from that of HIV and avian retroviruses in that it encodes an additional 50 amino acids N-terminal to the HHCC zinc-binding domain. This adds complexity to the NMR structural analysis of the 105 aa dimer. Through development of a novel growth/labeling system in E. coli utilizing condensed cultures, a preliminary NMR structure of the MuLV IN NTD monomer has been obtained. Experiments modify this system utilizing amino acid auxotroph strains to determine the dimer structure by NMR. Structural comparison with the IN NTD X-ray structure will be made, for which diffraction quality crystals have been obtained. SAXS analysis of the full-length IN ( DNA) will be performed. The IN NTD structures will be critical in defining domain localizations within the synaptic complex. Based on the structural analysis, the predictive function of the MuLV IN NTD will be analyzed. Specifically, the role of the putative winged-helix domain to interact with chromatinized DNA or associated factors will be determined. Mutagenesis studies will identify the role of specific amino acids in the dimer interface as well as interacting with viral and host factors. The ability of known DNA viral tethering domains to functionally complement the mutant IN NTD domains will be examined. Insights into the tethering of IN to chromatin has broad applications into target-site selection, but also to the requirement for mitosis for MuLV-based vectors. The safety of retroviral-based vectors is dependent on the target-site selection during retroviral integration. Through this understanding, mechanism to alter or limit integration can then be rationally designed.
Retroviral integration results in the stable incorporation of the viral DNA into the host chromosome and accounts for both the major benefit and shortfall of retroviral-based vectors. Understanding the determinants for target-site selection, and thus the potential for gene expression and/or disruption is key to developing safe vectors. This project analyzes the structure of the N-terminal domain of the Murine Leukemia Virus Integrase protein and that of the related XMRV virus. Structure-based modeling and viral studies are used to define its function in the assembly of the preinitegrative complex and/or tethering to DNA.
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