Plumpton M, Sharp N A, Liddicoat L H, Remm M, Tucker D O, Hughes F J, Russell S M, Romanos M A
Wellcome Research Laboratories, Beckenham, Kent, UK.
Biotechnology (N Y). 1995 Nov;13(11):1210-4. doi: 10.1038/nbt1195-1210.
The discovery of antiviral compounds against human papillomaviruses (HPV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication. However, plasmids containing the HPV replication origin replicate transiently upon co-transfection with HPV E1 and E2 expression vectors. We have adapted this assay using secreted alkaline phosphatase (SAP) as a reporter for rapid analysis of DNA copy number. Use of the SV40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy number dependence: the stronger beta-actin promotor inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP. The precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replication and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfections; under optimal conditions SAP increased 100-fold during replication. The assay has been developed for compound screening in 96-well plates and several inhibitors have been identified. Quantitative Southern blot analysis has shown that most of these inhibit HPV DNA replication rather than SAP accumulation or activity, and several are under test in models of viral replication. The assay also provides a rapid system for functional analysis of the HPV E1, E2 genes and the replication origin.
针对人乳头瘤病毒（HPV）的抗病毒化合物的发现一直受到体外培养病毒或检测稳定的HPV DNA复制的困难所阻碍。然而，含有HPV复制起点的质粒在与HPV E1和E2表达载体共转染时会瞬时复制。我们采用分泌碱性磷酸酶（SAP）作为报告基因对该检测方法进行了改进，用于快速分析DNA拷贝数。使用SV40早期启动子控制SAP表达对于确保强信号和拷贝数依赖性至关重要：较强的β-肌动蛋白启动子会抑制复制，而较弱的SV40晚期启动子产生的SAP水平非常低。E1和E2表达载体的精确配置也很关键，大多数现有的载体不支持高效复制和SAP分泌。DNA复制程度和SAP分泌均与转染中使用的E1/E2载体量成正比；在最佳条件下，复制过程中SAP增加100倍。该检测方法已开发用于96孔板中的化合物筛选，并已鉴定出几种抑制剂。定量Southern印迹分析表明，其中大多数抑制HPV DNA复制而非SAP积累或活性，有几种正在病毒复制模型中进行测试。该检测方法还为HPV E1、E2基因和复制起点的功能分析提供了一个快速系统。
