Integration of Murine Retroviral Vectors

描述(由申请人提供)：尽管鼠源性逆转录病毒与致癌转化和插入突变有关，但仍在作为病毒载体发展。在过去的一个月里，随着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，这一点得到了强调。尽管对整合部位的逆转录病毒/慢病毒的比较分析已经注意到宿主染色体内定位的差异，但将鼠源性病毒拴在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。这项建议发展和扩展了Moloney小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，以确定该结构域在前整合复合体中的功能。对该结构域的结构分析将扩展到XMRV，它与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，它编码HCC锌结合结构域的另外50个氨基酸。这增加了105氨基酸二聚体的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建立一种新的生长/标记系统，初步获得了MuLV IN NTD单体的核磁共振结构。实验利用氨基酸营养缺陷菌对该体系进行了改进，通过核磁共振确定了二聚体的结构。与In NTD X射线结构进行了结构比较，得到了具有衍射性质的晶体。将进行全长IN(DNA)的SAXS分析。IN NTD结构在定义突触复合体内的结构域定位方面将是至关重要的。在结构分析的基础上，分析了NTD中MULV的预测功能。具体地说，假定的翼状螺旋结构域与染色质DNA或相关因子相互作用的作用将被确定。突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。已知的DNA病毒拴系结构域在功能上补充突变的NTD结构域的能力将被检测。对IN与染色质捆绑的见解在靶点选择以及基于MuLV的载体的有丝分裂要求方面都有广泛的应用。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点的选择。通过这种理解，可以合理地设计改变或限制一体化的机制。 公共卫生相关性：逆转录病毒整合导致病毒DNA稳定地并入宿主染色体，这是逆转录病毒载体的主要优点和不足之处。了解靶点选择的决定因素，从而了解基因表达和/或破坏的可能性，是开发安全载体的关键。本项目分析了小鼠白血病病毒整合酶蛋白和相关XMRV病毒的N-末端结构域。基于结构的建模和病毒研究被用来确定它在前转录复合体组装和/或与DNA的连接中的功能。

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.