Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.
Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.
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稳定的细胞种群的产生和表征以诱导方式释放基于HIV-1的病毒（如颗粒）。
DOI：
10.1186/1472-6750-6-52
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发表时间：
2006-12-27
影响因子：
3.5
通讯作者：
Federico, Maurizio
中科院分区：
文献类型：
--
作者：
Muratori, Claudia;D'Aloja, Paola;Superti, Fabiana;Tinari, Antonella;Sol-Foulon, Nathalie;Sparacio, Sandra;Bosch, Valerie;Schwartz, Olivier;Federico, Maurizio
释放荧光病毒颗粒的细胞系的可用性可以显着支持各种研究，包括病毒-细胞相互作用的研究和抗病毒化合物的筛选。对于 HIV-1，由于许多 HIV-1 产品固有的细胞毒性，此类生物试剂的回收是一项非常艰巨的挑战。我们试图通过使用以诱导方式释放 HIV-1 颗粒的细胞系，并利用 HIV-1 Nef 突变体以相当高的水平掺入病毒颗粒的能力来克服这种限制。在这里，我们报告了 HIV-1 包装细胞系（称为 18-4s）的分离和表征，该细胞系能够以诱导方式释放大量基于荧光 HIV-1 的病毒样颗粒 (VLP)。通过在先前分离的诱导型 HIV-1 包装细胞系中组成型表达与增强型绿色荧光蛋白 (NefG3C-GFP) 融合的 HIV-1 NefG3C 突变体来回收 18-4s 细胞。 G3C 突变产生了棕榈酰化位点，导致 NefG3C-GFP 掺入病毒粒子的程度大大超过了野生型对应物。用松甾酮 A 和丁酸钠诱导 18-4s 细胞后，高达 4 μg/ml 的 VLP（每个病毒颗粒掺入了约 150 个 NefG3C-GFP 分子）被释放到培养物上清液中。由于其固有的强荧光，18-4s VLP 可以通过此处开发的新型细胞荧光测定法轻松检测到。用不同糖蛋白受体假型化的荧光 18-4 VLP 处理靶细胞，导致这些细胞在攻击后两小时就发出荧光。我们创建了一种稳定的细胞系，在诱导后释放基于 HIV-1 的荧光 VLP，可用于多种应用，包括病毒-细胞相互作用的研究和抗病毒化合物的筛选。
The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 μg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.
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影响因子：
5.4
作者：
Krautkrämer, E;Giese, SI;Fackler, OT
通讯作者：
Fackler, OT
影响因子：
2.2
作者：
Lamparski, HG;Metha-Damani, A;Le Pecq, JB
通讯作者：
Le Pecq, JB
影响因子：
5.4
作者：
Müller, B;Daecke, J;Kräusslich, HG
通讯作者：
Kräusslich, HG
影响因子：
5.4
作者：
Dull, T;Zufferey, R;Naldini, L
通讯作者：
Naldini, L
影响因子：
5.4
作者：
Fredericksen, BL;Wei, BL;Garcia, JV
通讯作者：
Garcia, JV