Hölscher C, Hörer M, Kleinschmidt J A, Zentgraf H, Bürkle A, Heilbronn R
Deutsches Krebsforschungszentrum, Angewandte Tumorvirolgie, Heidelberg, Germany.
J Virol. 1994 Nov;68(11):7169-77. doi: 10.1128/JVI.68.11.7169-7177.1994.
The adeno-associated virus (AAV) rep gene codes for a family of nonstructural proteins which are required for AAV gene regulation and DNA replication. In addition, rep has been implicated in a variety of activities outside the AAV life cycle which have been difficult to study, since attempts to achieve separate and constitutive expression of rep in stable cell lines have failed so far. Here we report the generation of two cell lines which inducibly express Rep78 under the control of the glucocorticoid-responsive mouse mammary tumor virus promoter. In addition, one of the cell lines constitutively expresses relatively high levels of Rep52. Both cell lines showed similar plating efficiencies with and without induction of Rep78 expression, which rules out cytotoxic effects of Rep78. The cell lines efficiently support DNA replication of a rep-negative AAV genome and initiate the formation of AAV particles. However, despite the correct sizes and stoichiometry of the three capsid proteins, the AAV particles were noninfectious. This was found to be due to a defect in the accumulation of single-stranded AAV DNA. Transient transfection of single expression constructs for constitutive, high-level expression of individual Rep proteins (either Rep78, Rep68, Rep52, or Rep40) complemented this defect. Infectious rep-negative AAV progeny was produced at varying efficiencies depending on the rep expression construct used. These data show that functional expression of full-length Rep in recombinant cell lines is possible and that the state of Rep expression is critical for the infectivity of AAV progeny produced.
腺相关病毒（AAV）的rep基因编码一类非结构蛋白，这些蛋白是AAV基因调控和DNA复制所必需的。此外，rep还参与了AAV生命周期之外的多种活动，而这些活动一直难以研究，因为迄今为止，在稳定细胞系中实现rep的单独组成型表达的尝试均告失败。在此，我们报告了两个细胞系的产生，它们在糖皮质激素应答性小鼠乳腺肿瘤病毒启动子的控制下可诱导表达Rep78。此外，其中一个细胞系组成型表达相对高水平的Rep52。无论是否诱导Rep78表达，这两个细胞系均显示出相似的铺板效率，这排除了Rep78的细胞毒性作用。这些细胞系有效地支持rep阴性AAV基因组的DNA复制，并启动AAV颗粒的形成。然而，尽管三种衣壳蛋白的大小和化学计量正确，但AAV颗粒却没有感染性。发现这是由于单链AAV DNA积累存在缺陷所致。瞬时转染用于组成型、高水平表达单个Rep蛋白（Rep78、Rep68、Rep52或Rep40）的单个表达构建体可弥补这一缺陷。根据所使用的rep表达构建体，产生具有感染性的rep阴性AAV子代的效率各不相同。这些数据表明，在重组细胞系中全长Rep的功能性表达是可能的，并且Rep的表达状态对于所产生的AAV子代的感染性至关重要。
