Assessment of a cytoprotection assay for the discovery and evaluation of anti-human immunodeficiency virus compounds utilizing a genetically-impaired virus
Assessment of a cytoprotection assay for the discovery and evaluation of anti-human immunodeficiency virus compounds utilizing a genetically-impaired virus
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DOI：
10.1016/0166-0934(95)01998-7
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发表时间：
1996-04-26
影响因子：
3.1
通讯作者：
Clanton, DJ
中科院分区：
文献类型：
--
作者：
Kiser, R;Makovsky, S;Clanton, DJ
一种生物包含的细胞保护试验被开发出来，以筛选人类免疫缺陷病毒的抑制剂，而不需要高水平的遏制或做法。所使用的病毒具有多个点突变，这些点突变破坏了其产生Rev和达特（病毒体外复制所必需的蛋白质）的能力。采用的原始细胞系（CEM-SSTART）含有允许Rev和达特连续表达的遗传构建体，并开发了提供最大急性细胞病变效应的亚克隆（1A 2）。国家癌症研究所的艾滋病药物筛选试验用于在T4淋巴细胞系CEM-SS中用HIVIIIB病毒和在1A 2亚克隆中用突变病毒来检测已知药物。这种基于细胞的测定使用四唑盐XTT作为细胞感染病毒后细胞代谢的指示剂。广泛测试的结果表明，使用突变病毒的检测方法与目前NCI艾滋病药物筛选方法相当。在1A 2或CEM-SS细胞培养物中培养42天后，病毒或整合的基因组没有恢复为野生型，并且在1A 2细胞中产生的病毒不能在PBMC中复制。突变病毒储备液不含野生型病毒，如通过PCR测定所确定的，该测定将发现60-600个突变RNA拷贝。这些材料，这是现在提供给科学界（美国国立卫生研究院艾滋病研究和参考试剂计划），应该是有用的工具，筛选和测试化合物的潜在抑制艾滋病毒在实验室没有配备维护和使用野生型传染性病毒。
A biologically contained cytoprotection assay was developed to screen inhibitors of the human immunodeficiency virus without the need for high level containment or practices. The virus used has multiple point mutations that have destroyed its ability to produce both Rev and Tat, proteins essential for virus replication in vitro. The original cell line employed (CEM-SSTART) contains a genetic construct that allows for the continuous expression of both Rev and Tat, and a subclone (1A2) was developed that provides for maximum acute cytopathic effect. The National Cancer Institute's AIDS drug screening assay was used to rest known drugs with both HIVIIIB virus in the T4 lymphocytic cell line CEM-SS and mutant virus in the 1A2 subclone. This cell-based assay uses the tetrazolium salt, XTT, as an indicator of cellular metabolism after the cells have been infected with virus. The results of extensive testing have shown that the assay using mutant virus is comparable to the current NCI AIDS drug screen. After 42 days in 1A2 or CEM-SS cell culture, the virus or the integrated genome did not revert to wild-type, and the virus produced in 1A2 cells was unable to replicate in PBMCs. Mutant viral stocks were devoid of wild-type virus as determined by a PCR assay that would have found 60-600 copies of mutant RNA. These materials, which are now available to the scientific community (NIH AIDS Research and Reference Reagent Program), should be useful tools to screen and test compounds for potential inhibition of HIV in laboratories not equipped to maintain and use wild-type infectious virus.