A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer.
A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer.
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DOI：
10.1016/j.jviromet.2005.04.012
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发表时间：
2005-09
影响因子：
3.1
通讯作者：
Mouland AJ
中科院分区：
文献类型：
--
作者：
Hu K;Clément JF;Abrahamyan L;Strebel K;Bouvier M;Kleiman L;Mouland AJ
本文介绍了一种灵敏的检测人类免疫缺陷病毒1型（HIV-1）蛋白酶（PR）活性的报告基因检测方法。该测定法测量PR活性作为供体分子[人源化海肾肾荧光素酶（hRLuc）]和能量受体分子，人源化绿色荧光蛋白（hGFP 2）在哺乳动物细胞中表达时的共振能量转移（RET）的函数。这是一种自然发生的现象，是一种新兴的强大技术，与替代的体外PR测定相比具有显著优势。将HIV-1 Gag-p2/Gag-p7（p2/p7）PR位点插入hGFP 2和hRLuc之间。新创建的载体hRLuc-p2/p7-hGFP 2与HIV-1密码子优化的PR+或PR− Gag/Pol表达子共表达。单独表达hRLuc-p2/p7-hGFP 2或与PR− Gag-Pol表达子一起表达产生BRET 2，表明PR切割位点未被切割，而包含PR+ Gag-Pol产生BRET 2的显著减少。PR抑制剂沙奎那韦或安普那韦的加入，或p2/p7 PR底物突变体的表达也阻断了切割，从而产生稳定的BRET 2信号。由于HIV-1辅助蛋白Vif已显示调节HIV-1 p2/p7切割，因此随后在表达Vif的研究中验证了该测定。当Vif与hRLuc-p2/p7-hGFP 2和PR+ Gag-Pol一起沿着过表达时，BRET 2的降低以剂量依赖性方式消除，表明超生理水平的Vif阻断p2/p7切割。观察到Gag加工中间体的积累，表明p2/p7切割受到负面影响。过表达的RNA结合缺陷的施陶芬蛋白或相关的dsRNA结合蛋白TRBP没有PR切割活性的影响所示的Western和BRET 2分析。p2/p7加工数据通过蛋白质印迹分析确认。BRET是非侵入性的，并且发生在活细胞内，在真实的时间内测量，并且不限于细胞区室，使其成为鉴定小的生物活性抑制分子的特别有吸引力的技术。该PR BRET 2生物传感器测定可适用于新的HIV-1 PR抑制剂的高通量筛选。它可用于筛选也靶向其他病毒蛋白酶的抗病毒化合物。
A sensitive reporter assay to measure human immunodeficiency virus type 1 (HIV-1) protease (PR) activity is described in this manuscript. This assay measures PR activity as a function of the resonance energy transfer (RET) between a donour molecule [humanized sea pansy Renilla reniformis luciferase (hRLuc)] and an energy acceptor molecule, humanized green fluorescent protein (hGFP2) when expressed in mammalian cells. This is a naturally occurring phenomenon and is an emerging and powerful technology that has significant advantages over alternative in vitro PR assays. The HIV-1 Gag-p2/Gag-p7 (p2/p7) PR site was inserted between hGFP2 and hRLuc. The newly created vector, hRLuc-p2/p7-hGFP2 was co-expressed with an HIV-1 codon-optimized PR+ or PR− Gag/Pol expressor. Expression of the hRLuc-p2/p7-hGFP2 alone or with the PR− Gag-Pol expressor generated a BRET2 indicating that the PR cleavage site was not cleaved, whereas the inclusion of the PR+ Gag-Pol produced a significant reduction in the BRET2. The inclusion of PR inhibitors Saquinavir or Amprenavir, or the expression of a p2/p7 PR substrate mutant also blocked the cleavage to result in a stable BRET2 signal. Because the HIV-1 auxiliary protein Vif has been shown to modulate the HIV-1 p2/p7 cleavage, this assay was then validated in studies in which Vif was expressed. When Vif was overexpressed along with hRLuc-p2/p7-hGFP2 and PR+ Gag-Pol, the decrease in BRET2 was abrogated in a dose-dependent manner, demonstrating that supraphysiologic levels of Vif block p2/p7 cleavage. An accumulation of a Gag processing intermediate was observed, indicating that p2/p7 cleavage was negatively affected. Overexpression of an RNA-binding-defective Staufen protein or a related dsRNA-binding protein TRBP had no effect on PR cleavage activity as shown by Western and BRET2 analyses. The p2/p7 processing data were confirmed by Western blot analyses. BRET is non-invasive and occurs within live cells, is measured in real time, and is not restricted to cellular compartments making it an especially attractive technology to identify small bioactive inhibitory molecules. This PR BRET2 biosensor assay can be adapted for high throughput screening of new HIV-1 PR inhibitors. It can be employed to screen for antiviral compounds that also target the proteases of other viruses.
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