Hölscher C, Kleinschmidt J A, Bürkle A
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
J Virol. 1995 Nov;69(11):6880-5. doi: 10.1128/JVI.69.11.6880-6885.1995.
Adeno-associated virus (AAV) codes for four closely related nonstructural proteins (Rep) required for AAV DNA replication and gene regulation. In vitro studies have revealed that either Rep78 or Rep68 alone is sufficient for AAV DNA replication. Rep52 and Rep40 are not required for DNA replication but have been reported to enhance the efficiency of accumulation of single-stranded progeny DNA. Previous studies on rep-expressing cell lines had indicated that only a subset of the four Rep proteins are required for the production of infectious AAV. We therefore set out to determine the minimal set of Rep proteins sufficient for the generation of infectious AAV. Transient cotransfections in HeLa cells of constructs for high-level expression of individual Rep proteins with a rep-negative AAV genome revealed that either Rep78 or Rep68 alone could complement for a full replication cycle yielding infectious virus. This result was confirmed by transfection studies in the cell line HeM2, which selectively expresses Rep78 at rather low levels under the control of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat (C. Hölscher, M. Hörer, J. A. Kleinschmidt, H. Zentgraf, A. Bürkle, and R. Heilbronn, J. Virol. 68:7169-7177, 1994). Increasing the level of Rep78 expression by transfection of a glucocorticoid receptor expression construct resulted in a higher level of DNA replication of a cotransfected rep-negative AAV genome and in the production of infectious rep-negative AAV particles. We further report on the generation of a new rep-expressing cell line, HeCM1, which was obtained by stable supertransfection of a construct for constitutive Rep40 expression into HeM1 cells (Hölscher et al., J. Virol. 68:7169-7177). Transfection of rather large amounts of rep-negative AAV DNA led to detectable virus production in HeCM1 cells even in the absence of the cotransfected glucocorticoid receptor expression construct, but higher yields were obtained after increasing the Rep78 level by coexpression of the glucocorticoid receptor. These data demonstrate that all Rep functions required for the productive replication of AAV in HeLa cells are contained within both Rep78 and Rep68.
腺相关病毒（AAV）编码四种与AAV DNA复制和基因调控所需的紧密相关的非结构蛋白（Rep）。体外研究表明，单独的Rep78或Rep68足以进行AAV DNA复制。DNA复制不需要Rep52和Rep40，但据报道它们可提高单链子代DNA的积累效率。先前对表达rep的细胞系的研究表明，四种Rep蛋白中只有一个子集是产生感染性AAV所必需的。因此，我们着手确定足以产生感染性AAV的最小Rep蛋白集。在HeLa细胞中，将用于单个Rep蛋白高水平表达的构建体与rep阴性AAV基因组进行瞬时共转染，结果表明，单独的Rep78或Rep68都可以补充完整的复制周期，产生感染性病毒。在细胞系HeM2中进行的转染研究证实了这一结果，该细胞系在糖皮质激素反应性小鼠乳腺肿瘤病毒长末端重复序列的控制下选择性地以相当低的水平表达Rep78（C. Hölscher、M. Hörer、J. A. Kleinschmidt、H. Zentgraf、A. Bürkle和R. Heilbronn，《病毒学杂志》68:7169 - 7177，1994年）。通过转染糖皮质激素受体表达构建体来提高Rep78的表达水平，导致共转染的rep阴性AAV基因组的DNA复制水平更高，并产生感染性rep阴性AAV颗粒。我们还报告了一种新的表达rep的细胞系HeCM1的产生，它是通过将组成型Rep40表达构建体稳定超转染到HeM1细胞中获得的（Hölscher等人，《病毒学杂志》68:7169 - 7177）。即使在没有共转染的糖皮质激素受体表达构建体的情况下，向HeCM1细胞中转染大量的rep阴性AAV DNA也能导致可检测到的病毒产生，但通过共表达糖皮质激素受体提高Rep78水平后可获得更高的产量。这些数据表明，HeLa细胞中AAV有效复制所需的所有Rep功能都包含在Rep78和Rep68中。
