A new reporter cell line to monitor HIV infection and drug susceptibility in vitro
A new reporter cell line to monitor HIV infection and drug susceptibility in vitro
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DOI：
10.1073/pnas.94.9.4653
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发表时间：
1997-04-29
影响因子：
11.1
通讯作者：
Corbeil, J
中科院分区：
文献类型：
--
作者：
Gervaix, A;West, D;Corbeil, J
通过监测 p24 抗原产生的减少来确定体外 HIV 感染性及其抗逆转录病毒药物的抑制作用是昂贵且耗时的。此类测定也不允许随着时间的推移准确定量受感染细胞的数量。为了开发一种简单、快速和直接的方法来监测HIV感染，我们生成了稳定的T细胞系（GEM），其含有编码由HIV-1长末端重复驱动的绿色荧光蛋白（人源化S65T GFP）的质粒，选择了显示低本构背景荧光但在感染后显示高水平GFP表达的克隆。 根据荧光显微镜、细胞荧光测定法和流式细胞术的监测，在感染 HIV 后，HIV-1 感染在 2 至 4 天内诱导细胞相对荧光增加 100 至 1,000 倍。在不同感染复数中添加逆转录酶、蛋白酶和其他靶标的抑制剂可以准确确定药物敏感性，该技术还可以通过评估第一轮感染中感染的细胞数量来定量病毒制剂的感染性。总之，CEM-GFP报告细胞系提供了一种简单、快速、直接的方法来监测HIV感染滴度和抗逆转录病毒药物 合胞体诱导菌株的敏感性。
Determination of HIV infectivity in vitro and its inhibition by antiretroviral drugs by monitoring reduction of production of p24 antigen is expensive and time consuming. Such assays also do not allow accurate quantitation of the number of infected cells over time, To develop a simple, rapid, and direct method for monitoring HIV infection, we generated a stable T-cell line (GEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-I long terminal repeat, Clones were selected that displayed low constitutive background fluorescence, but a high level of GFP expression upon infection with HIV, HIV-1 infection induced a 100- to 1,000-fold increase in relative fluorescence of cells over 2 to 4 days as monitored by fluorescence microscopy, cytofluorimetry, and flow cytometry. Addition of inhibitors of reverse transcriptase, protease, and other targets at different multiplicities of infection permitted the accurate determination of drug susceptibility, This technique also permitted quantitation of infectivity of viral preparations by assessment of number of cells infected in the first round of infection, In conclusion, the CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.