Integration of Murine Retroviral Vectors

描述（由申请方提供）：尽管与致癌转化和插入诱变相关，但基于鼠的逆转录病毒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系突出了这一点。虽然整合位点的比较逆转录病毒/慢病毒分析已经注意到宿主染色体内定位的差异，但将基于鼠的病毒拴系到宿主染色体的机制尚不清楚。蛋白质的结构分析可以提供对功能的重要见解。该建议开发并扩展了莫洛尼鼠白血病病毒整合酶（M-MuLV IN）蛋白的N-末端结构域（NTD）的初步结构分析，以定义该结构域在前整合复合物中的功能。该结构域的结构分析将扩展到XMRV，其与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其编码HHCC锌结合结构域N末端的另外50个氨基酸。这增加了105 aa二聚体的NMR结构分析的复杂性。通过在大肠杆菌中开发一种新的生长/标记系统。利用浓缩培养物，获得了MuLV IN NTD单体的初步NMR结构。实验利用氨基酸营养缺陷型菌株改进该系统，通过NMR确定二聚体结构。将与IN NTD X射线结构进行结构比较，其中已经获得了衍射质量的晶体。将对全长IN（DNA）进行SAXS分析。IN NTD结构在定义突触复合物内的结构域定位中将是关键的。基于结构分析，将分析MuLV IN NTD的预测功能。具体地，将确定推定的翼状螺旋结构域与染色质化DNA或相关因子相互作用的作用。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。将检查已知DNA病毒系链结构域功能性地补充突变体IN NTD结构域的能力。深入了解IN与染色质的拴系在靶位点选择中具有广泛的应用，但也适用于基于MuLV的载体的有丝分裂的要求。基于逆转录病毒的载体的安全性取决于逆转录病毒整合过程中的靶位点选择。通过这种理解，可以合理地设计改变或限制集成的机制。 公共卫生相关性：逆转录病毒整合导致病毒DNA稳定地掺入宿主染色体中，并解释了基于逆转录病毒的载体的主要益处和不足。了解靶位点选择的决定因素，从而了解基因表达和/或破坏的潜力是开发安全载体的关键。本计画分析小鼠白血病病毒整合酶蛋白质及相关XMRV病毒之N端结构域。基于结构的建模和病毒研究被用来定义其功能的组装preinitegrative复合物和/或拴系到DNA。

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.