Investigating the intercellular spreading properties of the foamy virus Gag protein.
Investigating the intercellular spreading properties of the foamy virus Gag protein.
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DOI：
10.1371/journal.pone.0031108
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Saïb A
中科院分区：
文献类型：
--
作者：
Tobaly-Tapiero J;Zamborlini A;Bittoun P;Saïb A
据报道，在细胞和病毒蛋白中发现的称为蛋白转导结构域(PTD)的小区域可以有效地穿越生物膜。在这里，我们发现原型泡沫病毒(PFV)的结构Gag蛋白显然能够在细胞之间移动，并将绿色荧光蛋白(GFP)从少数转基因细胞运送到整个单层的细胞核。缺失研究表明，这一特性存在于蛋白质C末端的第二个甘氨酸/精氨酸(GRII)框内。我们还发现，只有在甲醇固定后，受体细胞才能观察到表达为GFP-融合蛋白的Gag GRII的摄取和核积累，而在活细胞中，或者当细胞在甲醇固定之前用戊二醛固定或三氯乙酸处理时，则没有观察到。基于PFV长末端重复序列反式激活的灵敏荧光素酶活性分析进一步证实体内没有细胞间扩散。因此，我们得出结论，PFV Gag的细胞间传播代表了细胞固定过程中发生的人工扩散事件，随后是由Gag GRII盒内的染色质结合序列介导的核保留。根据这些结果，我们建议在根据荧光显微镜观察到的细胞间扩散的基础上将肽定义为PTD之前要谨慎。
Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.
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