An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.
An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.
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具有综合能力的诱导型细胞融合系统，可测量HIV-1进入抑制剂的效率和特异性。
DOI：
10.1371/journal.pone.0026731
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Sodroski JG
中科院分区：
文献类型：
--
作者：
Herschhorn A;Finzi A;Jones DM;Courter JR;Sugawara A;Smith AB 3rd;Sodroski JG
HIV-1包膜糖蛋白（Envs）通过融合病毒和靶细胞膜介导病毒进入，这是一个多步骤的过程，代表了一个有吸引力的抑制靶点。对不同的HIV-1分离株具有广泛活性的进入抑制剂可能作为开发治疗或预防性杀微生物剂的先导化合物非常有用。为了便于识别这样的抑制剂，我们已经构建了一个细胞-细胞融合系统，能够同时监测抑制效率和特异性。在该系统中，效应细胞稳定地表达四环素控制的反式激活因子（tTA），其能够紧密诱导HIV-1 Env和海肾荧光素酶（R-Luc）报告蛋白的表达。靶细胞表达HIV-1受体、CD4和CCR5，并携带在tTA响应启动子控制下的萤火虫荧光素酶（F-Luc）报告基因。因此，Env介导的这两种细胞类型的融合允许tTA扩散到靶细胞并激活F-Luc蛋白的表达。抑制剂阻断细胞-细胞融合的效率通过F-Luc活性的降低来测量，而抑制剂的特异性通过其对R-Luc活性的影响来评估。该系统表现出高动态范围和高Z '因子值。使用靶向HIV-1进入过程中不同步骤的抑制剂参考组验证了该测定法，产生的抑制浓度与已发表的病毒抑制数据相当。我们的系统适用于化学库的大规模筛选，也可用于已知进入抑制剂的抑制和细胞毒性特性的详细表征。
HIV-1 envelope glycoproteins (Envs) mediate virus entry by fusing the viral and target cell membranes, a multi-step process that represents an attractive target for inhibition. Entry inhibitors with broad-range activity against diverse isolates of HIV-1 may be extremely useful as lead compounds for the development of therapies or prophylactic microbicides. To facilitate the identification of such inhibitors, we have constructed a cell-cell fusion system capable of simultaneously monitoring inhibition efficiency and specificity. In this system, effector cells stably express a tetracycline-controlled transactivator (tTA) that enables tightly inducible expression of both HIV-1 Env and the Renilla luciferase (R-Luc) reporter protein. Target cells express the HIV-1 receptors, CD4 and CCR5, and carry the firefly luciferase (F-Luc) reporter gene under the control of a tTA-responsive promoter. Thus, Env-mediated fusion of these two cell types allows the tTA to diffuse to the target cell and activate the expression of the F-Luc protein. The efficiency with which an inhibitor blocks cell-cell fusion is measured by a decrease in the F-Luc activity, while the specificity of the inhibitor is evaluated by its effect on the R-Luc activity. The system exhibited a high dynamic range and high Z'-factor values. The assay was validated with a reference panel of inhibitors that target different steps in HIV-1 entry, yielding inhibitory concentrations comparable to published virus inhibition data. Our system is suitable for large-scale screening of chemical libraries and can also be used for detailed characterization of inhibitory and cytotoxic properties of known entry inhibitors.
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影响因子：
56.9
作者：
Feng, Y;Broder, CC;Berger, EA
通讯作者：
Berger, EA
影响因子：
4.8
作者：
Garcia-Perez, Javier;Rueda, Patricia;Kellenberger, Esther
通讯作者：
Kellenberger, Esther
影响因子：
56.9
作者：
BARRESINOUSSI, F;CHERMANN, JC;MONTAGNIER, L
通讯作者：
MONTAGNIER, L
影响因子：
5.4
作者：
Guo, Q;Ho, HT;Lin, PF
通讯作者：
Lin, PF
DOI：
10.1097/hjr.0b013e328336a150
发表时间：
2010-10-01
影响因子：
--
作者：
Friis-Moller, Nina;Thiebaut, Rodolphe;Law, Matthew G.
通讯作者：
Law, Matthew G.