Replication of BK DNA in Murine Cells and Extracts

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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.