Ling J Y, Kienzle T E, Chen T M, Henkel J S, Wright G C, Stroop W G
Division of Molecular Virology, Baylor College of Medicine, Houston, TX, USA.
Virus Res. 1997 Jul;50(1):95-106. doi: 10.1016/s0168-1702(97)00038-5.
We have analyzed the activity of a specific portion of the latency-associated transcript (LAT) promoter of three strains of herpes simplex virus type 1 (HSV-1) in neuronal and non-neuronal cell types. Restriction fragments containing the LAT promoter sequences and the 5'-end of the LATs were isolated from HSV-1 strains H129, +GC and KOS-63, sequenced and cloned into a chloramphenicol transferase (CAT) plasmid vector. These vectors were separately assayed for CAT production in human (SknSH) and mouse (C-1300) neuroblastoma cell lines and a human continuous cell line (HeLa). Strain KOS-63 contained a C to T base substitution within the LAT promoter binding factor element upstream of the cAMP response element binding sequence. In replicate experiments, in which the construct DNA was used for transfection, the CAT constructs from strains H129 and +GC functioned equally well in all three cell lines. In contrast, the strain KOS-63 CAT construct functioned significantly better in HeLa cells than in neuroblastoma cell lines and better than the identical CAT constructs from strains H129 and +GC. In addition, the construct from strain KOS-63 functioned less well in the human neuroblastoma cell line than in HeLa or C-1300 neuroblastoma cells. When LAT expression was examined directly in vivo by in situ hybridization, strain KOS-63 produced slightly less LAT RNA than strain H129 within trigeminal ganglionic neurons of latently infected rabbits. However, utilizing competitive gel-shift assays, DNA fragments containing the LAT promoter binding element from all three strains bound equivalent amounts of HeLa cell nuclear proteins. Together, these results suggest that the activity expressed by the strain KOS-63 LAT promoter in vivo and in vitro may relate to positive or negative effects of DNA binding proteins on LAT transcription, and that these effects are cell-type dependent.
我们分析了三株1型单纯疱疹病毒（HSV-1）潜伏相关转录物（LAT）启动子特定区域在神经元和非神经元细胞类型中的活性。从HSV-1毒株H129、+GC和KOS-63中分离出包含LAT启动子序列和LAT 5'端的限制性片段，进行测序并克隆到氯霉素转移酶（CAT）质粒载体中。分别检测这些载体在人神经母细胞瘤细胞系（SknSH）、小鼠神经母细胞瘤细胞系（C-1300）和人连续细胞系（HeLa）中CAT的产生情况。毒株KOS-63在cAMP反应元件结合序列上游的LAT启动子结合因子元件内存在一个C到T的碱基替换。在重复实验中，将构建体DNA用于转染，来自毒株H129和+GC的CAT构建体在所有三种细胞系中的功能相同。相比之下，毒株KOS-63的CAT构建体在HeLa细胞中的功能明显优于神经母细胞瘤细胞系，且优于来自毒株H129和+GC的相同CAT构建体。此外，毒株KOS-63的构建体在人神经母细胞瘤细胞系中的功能不如在HeLa或C-1300神经母细胞瘤细胞中。当通过原位杂交直接在体内检测LAT表达时，在潜伏感染兔子的三叉神经节神经元中，毒株KOS-63产生的LAT RNA略少于毒株H129。然而，利用竞争性凝胶迁移分析，来自所有三株毒株的含有LAT启动子结合元件的DNA片段结合等量的HeLa细胞核蛋白。这些结果共同表明毒株KOS-63的LAT启动子在体内和体外表达的活性可能与DNA结合蛋白对LAT转录的正向或负向作用有关，且这些作用具有细胞类型依赖性。
