Gervaix A, West D, Leoni L M, Richman D D, Wong-Staal F, Corbeil J
Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0679, USA.
Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4653-8. doi: 10.1073/pnas.94.9.4653.
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 (CEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-1 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.
通过监测p24抗原产生的减少来测定体外HIV感染性及其被抗逆转录病毒药物抑制的情况既昂贵又耗时。此类检测也无法随时间准确定量受感染细胞的数量。为了开发一种简单、快速且直接的监测HIV感染的方法，我们构建了一个稳定的T细胞系（CEM），该细胞系含有一个由HIV-1长末端重复序列驱动的编码绿色荧光蛋白（人源化S65T GFP）的质粒。筛选出的克隆显示出低组成性背景荧光，但在感染HIV后GFP表达水平较高。通过荧光显微镜、细胞荧光测定法和流式细胞术监测，HIV-1感染在2至4天内使细胞的相对荧光增加了100至1000倍。在不同感染复数下添加逆转录酶、蛋白酶和其他靶点的抑制剂可准确测定药物敏感性。该技术还可通过评估第一轮感染中受感染细胞的数量来定量病毒制剂的感染性。总之，CEM-GFP报告细胞系为监测合胞体诱导株的HIV感染滴度和抗逆转录病毒药物敏感性提供了一种简单、快速且直接的方法。
