Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity
Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity
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DOI：
10.1128/aac.45.9.2616-2622.2001
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发表时间：
2001-09-01
影响因子：
4.9
通讯作者：
Dantuma, NP
中科院分区：
文献类型：
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作者：
Lindsten, K;Uhlíková, T;Dantuma, NP
人类免疫缺陷病毒1型（HIV-1）蛋白酶是产生感染性病毒所必需的，因此是开发抗艾滋病药物的主要目标。细胞蛋白质也被蛋白酶切割，这解释了其细胞毒性活性以及随后未能建立方便的基于细胞的蛋白酶测定。我们已经利用这种毒性开发了一种新的蛋白酶测定法，该测定法依赖于携带绿色荧光蛋白（GFP-PR）的人工蛋白酶前体的瞬时表达。前体在体内通过自催化裂解被激活，导致蛋白酶表达细胞的快速消除。用治疗剂量的HIV-1蛋白酶抑制剂治疗导致荧光前体的剂量依赖性积累，其可以通过流式细胞术和荧光测定法容易地检测和定量。前体提供了一个方便和非感染性的模型，用于高通量筛选可以干扰活细胞中蛋白酶活性的物质。
The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-througput screenings of substances that can interfere with the activity of the protease in living cells.