REGULATION OF AAV DNA REPLICATION

腺相关病毒(AAV)/猴病毒40(SV40)混合型病毒 通过插入SV40调节基因构建了基因组 将AAV基因组(5171-5243-270)插入缺失。 NT 144-264。删除会移除最左侧的AAV 启动子和上游约100个碱基，但在 杂交基因组从缺失的转录本的帽子位置 AAV启动子。插入的序列包含SV40原点 复制(ORI)。然而，当被转染到细胞中时， 原始性表达SV40T抗原的质粒复制 很差。我们已经发现了复制的抑制作用 需要来自AAV rep基因的反式作用产物(开放的 基因组左半部分的读框，其产物是 复制所必需的)和两个顺式作用靶序列 它们位于AAV的反向终端中继器内 基因组。这项建议描述了细胞培养和 体外研究这种负性反应的机制 监管。在细胞培养实验中，我们将确定：1) 目标序列的准确参数，2)距离是否 SV40 ORI和靶序列之间的关系很重要，3) 是否存在ORIS与靶序列的临界比例，4) 是否可以被过量的T抗原克服抑制，5) 部分AAV rep基因编码抑制产物和6) 病毒癌基因表达能否克服抑制， 或用物理或化学致癌物治疗细胞。我们 计划使用已建立的SV40DNA体外检测方法 复制作为抑制物分离过程中的一种分析方法 受感染细胞的活性。描述了分离的实验 AAV rep基因产物来自受感染的细胞或由 表达载体的方法。还将有一种尝试 体外合成具有活性的rep基因产物。通过这些手段，我们 希望能深入了解负面情绪背后的机制 AAV DNA复制的调控。

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.