Replication of BK DNA in Murine Cells and Extracts

描述(由申请人提供)：BK病毒很可能是多瘤病毒相关性肾病(PVAN)的主要病原体，可导致高达5%的肾移植失败。PVAN以病毒诱导的肾小管上皮细胞病理改变为特征，继之以混合性炎性间质浸润和小管炎为主，最终导致晚期肾小管间质纤维化。这些事件如何与BK重新激活和复制有关目前尚不清楚，可以通过合适的小鼠模型研究来回答。本研究的目的是提供有关BK病毒复制的基本信息，并为建立PVAN小鼠模型奠定基础。将检验两个假设：1)BK病毒的复制需要大T抗原和宿主蛋白(S)之间的功能性相互作用，这是核心复制复合体活动所必需的，这在小鼠细胞中是不存在的。将用纯化的人蛋白和小鼠细胞提取物研究BK的复制，并确定关键人类蛋白对BK复制的重要性；2)BK病毒在小鼠细胞中复制的限制不是由T抗原与核心复制蛋白的大范围相互作用决定的，而是由核心起源以外的序列(例如，早期和晚期区域/增强子非编码调控序列)和相关宿主蛋白决定的。将构建含有BK病毒增强子/非编码调控序列(mPy/bke)的嵌合小鼠多瘤病毒，或含有mPy增强子/非编码调控序列(bk/mpye)的嵌合BK病毒，并确定它们对人和小鼠肾脏细胞中BK基因表达和DNA复制的重要性。类固醇和其他非编码调控/增强子序列的调节剂将被用来探索非编码调控/增强子序列对BK和嵌合病毒DNA复制和基因表达的重要性。有了这一关于BK复制的新信息，可能会理解BK病毒在肾脏细胞中重新激活的分子机制，并可能建立PVAN的小鼠模型。远期目标是降低PVAN的发病率和发病率。

DESCRIPTION (provided by applicant): BK virus is likely to be the primary etiologic agent of polyomavirus associated nephropathy (PVAN) that causes allograft failure in up to 5% of renal transplants. PVAN is marked by viral-induced cytopathology in renal tubular cells followed by mixed inflammatory interstitial infiltrates and tubulitis, finally leading to interstitia fibrosis in late stage disease. How these events relate to BK reactivation and replication is presently unclear and could be answered by research in a suitable murine model. Goals of the proposed research are to develop basic information about the replication of BK virus, and to lay the foundation for the development of a murine model of PVAN. Two hypotheses will be tested: 1) Replication of BK virus requires functional interactions between the large T-antigen and host protein(s) required for activity of the core replication complex, and which do not occur in murine cells. The replication of BK will be studied with purified human proteins and murine cell extracts, and the importance of key human proteins for BK replication determined; 2) The restriction of BK virus replication in murine cells is determined not by large T-antigen interactions with the core replication proteins, but by sequences outside the core origin (e.g. the early and late region/enhancer non coding regulatory sequences) and associated host proteins. Chimeric murine polyomaviruses containing the BK virus enhancer/non coding regulatory sequences (mPy/BKe), or chimeric BK viruses containing the mPy enhancer/non-coding regulatory sequences (BK/mPye), will be constructed and their importance for BK gene expression and DNA replication defined in human and murine kidney cells. Steroid and other modulators of the non-coding regulatory/enhancer sequences will be used to probe the importance of the non-coding regulatory/enhancer sequences upon DNA replication and gene expression of BK and the chimeric viruses. With this new information about BK replication, molecular mechanisms responsible for the reactivation of BK virus in kidney cells may be understood, and murine models of PVAN may be made possible. The long-term goal is the reduction of the incidence and morbidity of PVAN.