CHARACTERIZATION OF ADENO ASSOCIATED VIRUS NON-STRUCTURAL PROTEINS

Rep与细胞蛋白的相互作用。过度表达较大Rep多肽的细胞具有独特的表型。这些细胞似乎在细胞周期中受阻，无法进行有丝分裂和细胞凋亡。最近，我们已经证明了Rep蛋白的非剪接形式Rep 78和Rep 52结合并抑制了环AMP反应细胞激酶PrKX和PKA。然而，Rep 78的剪接形式Rep 68保留了诱导凋亡的活性，尽管水平降低。因此，Rep蛋白可能通过多种活性的累积效应诱导细胞死亡，例如Rep 68和Rep 78的内切酶活性可能不是细胞凋亡所必需的。通过评估Rep蛋白的不同突变形式，可以分离出诱导细胞死亡所需的生化活性。Rep蛋白的生化活性。我们最近鉴定了Rep蛋白的特征，这可能为解释AAV生命周期中仍不清楚的方面提供基础。所有四种Rep蛋白都共享ATP结合口袋。我们已经证明，两个较小的Rep蛋白缺乏包含在两个较大的Rep蛋白中的特定DNA结合域，能够作为DNA解旋酶发挥作用。这与这些Rep蛋白在将单链DNA基因组包装到组装的衣壳中的假定作用是一致的。最近，我们已经证明了P5衍生的Rep蛋白作为DNA划痕和重新连接酶发挥功能。因此，这些蛋白可以正式定义为拓扑异构酶。所有单链DNA基因组都通过复制启动子蛋白进行复制，这些复制启动子蛋白的功能相似，与系统发育分布无关。例如，噬菌体M13，植物的双生病毒，或细小病毒科，编码一种蛋白质，识别DNA合成的同源来源。复制蛋白结合到原点，划出一条链，允许DNA聚合酶启动先导链DNA合成。因此，AAV Rep蛋白包含原核生物和植物复制蛋白所描述的活性。AAV Rep蛋白特异性地切割并重新连接DNA的能力可能直接参与AAV DNA复制以及AAV与人类19号染色体的定向整合。-腺相关病毒，AAV，复制启动蛋白

Rep interaction with cellular proteins. Cells which over-express the larger Rep polypeptides have a distinctive phenotype. These cells appear to be blocked in the cell cycle and are unable to undergo mitosis and undergo apoptotic cell death. Recently we have demonstrated that the unspliced forms of the Rep proteins, Rep 78 and Rep 52, bind and inhibit the cyclic AMP responsive cellular kinases, PrKX and PKA. However, the spliced form of Rep 78, Rep 68, retains the apoptotic inducing activities, although at a reduced level. Thus, it appears that Rep proteins induce apoptotic cell death by accumulated effects due to various activities, e.g. the endonuclease activity of Rep 68 and Rep 78 may not be necessary for apoptosis. By evaluating different mutated forms of the Rep proteins, the biochemical activities required for inducing cell death can be isolated. Biochemical activities of Rep proteins. We have recently characterized features of the Rep proteins that may provide a basis for explaining the aspects of AAV life-cycle that remained obscure. All four of the Rep proteins share the ATP binding pocket. We have shown that the two smaller Rep proteins which lack the specific DNA binding domain contained in the two larger Rep proteins, are able to function as DNA helicases. This is consistent with the putative role of these Rep proteins in packaging the single- stranded DNA genome into the assembled capsids. More recently, we have demonstrated that the p5 derived Rep proteins function as DNA nicking and re-joining enzymes. Thus, these proteins may be defined formally as toposiomerases. All single-stranded DNA genomes replicate through the use of replication initiator proteins that function similarly regardless of the phylogenetic distribution. For example, bacteriophage M13, geminivirus of plants, or parvoviridae, encodes a protein which recognizes the cognate origin of DNA synthesis. The replication protein binds to the origin, nicks one strand permitting a DNA polymerase to initiate leading-strand DNA synthesis. Thus, AAV Rep proteins contain the activities described for the prokaryotic and plant replication proteins. The ability of AAV Rep protein to specifically nick and rejoin DNA is likely to be involved directly in AAV DNA replication as well as targeted integration of AAV into human chromosome 19. - adeno- associated virus, AAV, replication initiator proteins