NEGATIVE REGULATION OF AAV DNA REPLICATION

腺相关病毒（AAV）/猿猴病毒40（SV 40）杂交 通过插入SV 40调节基因构建了基因组。 在AAV基因组中，将缺失区域（nt 5171-5243-270）插入来自 nt 144-264。 该删除移除最多的AAV 启动子和上游约100个碱基，但在 杂合基因组是来自缺失的杂合基因的转录物的帽位点。 AAV启动子。 插入的序列含有SV 40的起源， 复制（ori）。 然而，当转染到 组成型表达质粒复制的SV 40 T抗原 非常糟糕。 我们发现了复制的抑制 需要来自AAV rep基因的反式作用产物（开放的 阅读框位于基因组的左半部分，其产物是 复制所必需的）和两个顺式作用靶序列 其位于AAV的反向末端重复序列内 基因组 该提案描述了细胞培养实验， 在体外表征这种负的机制， 调控 在细胞培养实验中，我们将确定：1） 目标序列的精确参数，2）距离是否 SV 40 ori和靶序列之间的差异是重要的，3） 是否存在ORIS与靶序列的临界比率，4） 是否可以通过过量的T抗原克服抑制，5） AAV rep基因的一部分编码抑制产物，和6） 抑制是否可以被病毒癌基因表达所克服， 或用物理或化学致癌物处理细胞。 我们 计划使用已建立的SV 40 DNA体外试验 在抑制性药物的分级分离期间作为测定的复制 受感染细胞的活性。 实验被描述为分离 AAV rep基因产物来自受感染的细胞， 表达载体的方法。 还将尝试 体外合成活性REP基因产物。 通过这些手段，我们 我希望能深入了解这些负面影响的机制， 调节AAV DNA复制。

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome has been constructed by insertion of the SV40 regulatory region (nt 5171-5243-270) into a deletion in the AAV genome from nt 144-264. The deletion removes the leftward most AAV promoter and about 100 bases upstream, but leaves intact in the hybrid genome the cap site of the transcript from the deleted AAV promoter. The inserted sequence contains the SV40 origin of replication (ori). However, when transfected into cells that constitutively express the SV40 T-antigen the plasmid replicates very poorly. We have discovered the inhibition of replication requires a trans-acting product from the AAV rep gene (an open reading frame in the left half of the genome whose products are necessary for replication) and two cis-acting target sequences which are within the inverted terminal repeats of the AAV genome. This proposal describes experiments in cell culture and in vitro to characterize the mechanism of this negative regulation. In cell culture experiments we will determine: 1) the exact parameters of the target sequence, 2) whether the distance between SV40 ori and the target sequence is important, 3) whether there is a critical ratio of oris to target sequences, 4) whether inhibition can be overcome by excess T-antigen, 5) which part of the AAV rep gene encodes the inhibitory product and 6) whether inhibition can be overcome by viral oncogene expression, or treatment of cells with physical or chemical carcinogens. We plan to use the established in vitro assay for SV40 DNA replication as an assay during fractionation of the inhibitory activity from infected cells. Experiments are described to isolate that AAV rep gene products from either infected cells or by means of expression vectors. There will also be an attempt to synthesize active rep gene products in vitro. By these means we hope to gain insight into the mechanisms underlying the negative regulation of AAV DNA replication.