Roemer K, Johnson P A, Friedmann T
Center for Molecular Genetics, University of California, San Diego, La Jolla 92093.
J Virol. 1991 Dec;65(12):6900-12. doi: 10.1128/JVI.65.12.6900-6912.1991.
We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.
我们研究了一些影响整合到单纯疱疹病毒1型（HSV）中的外源启动子-报告基因构建体表达的参数。这些参数包括亲本病毒的遗传背景、转基因在HSV基因组中的插入位点以及受感染的细胞类型。标记为δ3的载体构建体的遗传背景是一种HSV 1型突变体，其几乎删除了必需的立即早期基因IE3的整个编码部分Vmw175（ICP4）的编码部分。对于标记为+的载体，通过标记拯救修复了IE3缺失。我们使用由猴病毒40（SV40）早期启动子和增强子区域驱动的细菌氯霉素乙酰转移酶（CAT）基因作为报告基因。将SV40-cat杂交基因插入HSV胸苷激酶（TK）基因座以构建载体TKScatδ3和TKScat+，或者插入病毒基因组短独特部分的BamHI z片段内的基因间位点以构建载体GScatδ3和GScat+。在用TKScatδ3感染的Vero和BHK细胞中，感染后10小时首次检测到CAT活性，并持续积累直至感染后36小时。在用具有复制能力的载体TKScat+感染的灵长类来源细胞中，或者在补充IE3缺陷并被TKScatδ3感染的灵长类细胞中，CAT活性明显低于啮齿类来源细胞。然而，在放线菌酮存在的情况下，CAT水平升高，这表明灵长类细胞中CAT产量低是由于SV40-cat杂交基因表达受到抑制。与TKScatδ3和TKScat+的结果相反，在用GScatδ3或GScat+感染的任何测试细胞类型中，除了在放线菌酮逆转的条件下，均未检测到CAT活性。这些结果表明，虽然在Vmw175存在下表达的HSV基因产物以细胞类型特异性方式抑制tk基因座中的SV40-cat表达，但在Vmw175存在或不存在的情况下表达的HSV基因产物独立于细胞类型抑制BamHI z基因座中的SV40-cat表达。
