Kool M, Voeten J T, Goldbach R W, Vlak J M
Department of Virology, Wageningen Agricultural University, The Netherlands.
Virology. 1994 Feb;198(2):680-9. doi: 10.1006/viro.1994.1080.
Previous results showed that plasmids containing one of the eight putative origins (ori's) of Autographa californica nuclear polyhedrosis virus (AcMNPV) are replicated after transfection into Spodoptera frugiperda cells if essential trans-acting factors are supplied by AcMNPV infection (Kool et al., Virology, 192, 94-101, 1993a; Kool et al., J. Gen. Virol., in press, 1993b; Leisy and Rohrmann, Virology, 196, 722-730, 1993). In this report a transient complementation assay is described in which four cotransfected cosmid clones, instead of AcMNPV infection, provided essential trans-acting factors for plasmid DNA replication. In this assay plasmid replication was found to be independent of the presence, in cis, of a viral ori. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay is a valid approach for identification of AcMNPV replication genes. We further used the assay to define essential regions in the four required cosmids. Six regions of the AcMNPV genome, EcoRI-I (map unit 0.3-5.8), EcoRI-O (map unit 6.9-8.7), SstI-F (map unit 38.9-45.0), EcoRI-D (map unit 59.9-68.3), a BamHI-SstII fragment of BamHI-B (map unit 84.3-89.7), and EcoRI-B (map unit 90.0-100), with at least seven genes, were found to be essential for plasmid DNA replication. These regions contain the putative DNA polymerase gene (SstI-F), the helicase-like gene (EcoRI-D), and the region where most of the trans-activating immediate--early genes of AcMNPV are located (EcoRI-B). For SstI-F it was shown that this region contains besides the DNA polymerase gene at least one other replication gene. These results show that it will now be possible to define the set of AcMNPV genes necessary and sufficient for DNA replication.
先前的结果表明，如果通过苜蓿银纹夜蛾核型多角体病毒（AcMNPV）感染提供必需的反式作用因子，那么含有苜蓿银纹夜蛾核型多角体病毒八个假定的复制起点（ori）之一的质粒转染到草地贪夜蛾细胞后会发生复制（库尔等人，《病毒学》，192，94 - 101，1993a；库尔等人，《普通病毒学杂志》，即将发表，1993b；利西和罗尔曼，《病毒学》，196，722 - 730，1993）。在本报告中描述了一种瞬时互补分析方法，其中四个共转染的黏粒克隆，而非AcMNPV感染，为质粒DNA复制提供必需的反式作用因子。在该分析中发现质粒复制不依赖于病毒ori的顺式存在。当从转染混合物中省略其中一个黏粒时，质粒不发生复制。这一结果表明该分析是鉴定AcMNPV复制基因的有效方法。我们进一步利用该分析来确定四个必需黏粒中的必需区域。发现AcMNPV基因组的六个区域，即EcoRI - I（图谱单位0.3 - 5.8）、EcoRI - O（图谱单位6.9 - 8.7）、SstI - F（图谱单位38.9 - 45.0）、EcoRI - D（图谱单位59.9 - 68.3）、BamHI - B的一个BamHI - SstII片段（图谱单位84.3 - 89.7）和EcoRI - B（图谱单位90.0 - 100），至少包含七个基因，对质粒DNA复制是必需的。这些区域包含假定的DNA聚合酶基因（SstI - F）、解旋酶样基因（EcoRI - D）以及AcMNPV大多数反式激活即刻早期基因所在的区域（EcoRI - B）。对于SstI - F，已表明该区域除了DNA聚合酶基因外还至少包含一个其他复制基因。这些结果表明现在有可能确定AcMNPV中DNA复制所必需且足够的基因集。
