Godeau F, Saucier C, Kourilsky P
Unité de Biologie Moléculaire du Gène, INSERM U 277, Institut Pasteur, Paris, France.
Nucleic Acids Res. 1992 Dec 11;20(23):6239-46. doi: 10.1093/nar/20.23.6239.
The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.
单纯疱疹病毒1型的胸苷-胸苷酸激酶（HSV1-TK）（ATP：胸苷5'-磷酸转移酶；EC 2.7.1.21）的表达赋予宿主细胞一种条件致死表型，该表型取决于核苷类似物的存在，这些核苷类似物被该酶代谢为DNA复制的毒性抑制剂。为了产生一种可被核苷类似物筛选的重组杆状病毒，将HSV1-tk编码序列置于苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的立即早期启动子IE-1(0)的控制下，并通过同源重组将该构建体引入AcMNPV的多角体蛋白基因座。分离出两种在多角体蛋白基因座携带该基因构建体的重组杆状病毒，并测试它们在不同浓度的核苷类似物9-(1,3-二羟基-2-丙氧甲基)鸟嘌呤（更昔洛韦）存在下的复制能力。浓度高达100μM的更昔洛韦对Sf9鳞翅目细胞活力以及野生型或表达β-半乳糖苷酶的重组AcMNPV的复制均无影响。相比之下，携带HSV1-tk基因的重组AcMNPV病毒的复制受到更昔洛韦的剂量依赖性抑制。在2μM时可检测到抑制作用，在100μM时完全抑制。在模型分离中利用了这一特性，旨在纯化由于病毒DNA与替代载体共转染后在多角体蛋白基因座发生同源重组而丢失了这种可反向选择基因标记的新重组病毒。在更昔洛韦存在下繁殖后，这种共转染的子代含有超过85%的重组病毒，表明更昔洛韦对含亲本HSV1-tk病毒的反向选择构成了一种分离重组杆状病毒的新方法。
