Integration of Murine Retroviral Vectors

描述（由申请人提供）：尽管基于小鼠的逆转录病毒与致癌转化和插入突变有关，但它们仍继续被开发为病毒载体。在过去的一个月里，异嗜性小鼠白血病病毒相关病毒（XMRV）与包括慢性疲劳综合征和前列腺癌在内的人类疾病的关联突出了这一点。虽然逆转录病毒/慢病毒对整合位点的比较分析已经注意到宿主染色体内定位的差异，但将小鼠病毒捆绑到宿主染色体上的机制尚不清楚。蛋白质的结构分析可以提供对功能的基本见解。本研究发展并扩展了Moloney小鼠白血病病毒整合酶（M-MuLV IN）蛋白n端结构域（NTD）的初步结构分析，以确定该结构域在预整合复合体中的功能。该结构域的结构分析将扩展到XMRV，与MuLV IN保持结构同源性。研究的第一个重点是IN NTD的结构研究。MuLV NTD与HIV和禽类逆转录病毒的不同之处在于，它在HHCC锌结合结构域上编码另外50个氨基酸n端。这增加了105aa二聚体核磁共振结构分析的复杂性。通过在大肠杆菌中利用浓缩培养物开发一种新的生长/标记系统，获得了MuLV in NTD单体的初步核磁共振结构。实验利用氨基酸营养不良菌株对该体系进行了修改，用核磁共振法测定了二聚体的结构。将与IN NTD x射线结构进行结构比较，获得了具有衍射质量的晶体。将进行全长IN （DNA）的SAXS分析。IN NTD结构对于确定突触复合体内的区域定位至关重要。在结构分析的基础上，分析了MuLV IN NTD的预测功能。具体来说，假定的翼状螺旋结构域与染色化DNA或相关因子相互作用的作用将被确定。诱变研究将确定特定氨基酸在二聚体界面中的作用以及与病毒和宿主因子的相互作用。我们将研究已知的DNA病毒捆绑结构域在功能上对突变的IN NTD结构域的补充能力。深入了解IN与染色质的结合在靶位点选择以及基于mulv的载体有丝分裂的要求方面具有广泛的应用。逆转录病毒载体的安全性取决于逆转录病毒整合过程中靶位点的选择。通过这种理解，就可以合理地设计改变或限制集成的机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.