Replication of BK DNA in Murine Cells and Extracts

描述（由申请人提供）：BK病毒可能是多瘤病毒相关肾病（PVAN）的主要病因，PVAN导致高达5%的肾移植失败。PVAN在肾小管细胞中表现为病毒诱导的细胞病理学，随后是混合炎性间质浸润和小管炎，最终在疾病晚期导致间质纤维化。目前尚不清楚这些事件与BK再激活和复制之间的关系，可以通过在合适的小鼠模型中进行研究来回答。本研究旨在了解BK病毒复制的基本信息，为建立小鼠PVAN模型奠定基础。两种假设将被验证：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性所必需的，而在小鼠细胞中不会发生。将用纯化的人蛋白和小鼠细胞提取物研究BK的复制，并确定关键人蛋白对BK复制的重要性；2)小鼠细胞中BK病毒复制的限制不是由大t抗原与核心复制蛋白的相互作用决定的，而是由核心起源外的序列（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/非编码调控序列的嵌合小鼠多瘤病毒（mPy/BKe），或含有BK病毒增强子/非编码调控序列的嵌合BK病毒（BK/mPye），以及它们对人类和小鼠肾细胞中BK基因表达和DNA复制的重要性。将利用非编码调控/增强子序列的类固醇和其他调节剂来探讨非编码调控/增强子序列对BK和嵌合病毒DNA复制和基因表达的重要性。有了这些关于BK复制的新信息，可能会了解肾细胞中BK病毒再激活的分子机制，并可能使小鼠PVAN模型成为可能。长期目标是减少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.