Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell
Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell
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DOI：
10.1016/j.virol.2006.10.025
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发表时间：
2007-04-10
期刊：
影响因子：
3.7
通讯作者：
Hope, Thomas J.
中科院分区：
文献类型：
--
作者：
Campbell, Edward M.;Perez, Omar;Hope, Thomas J.
GFP-VPR标记的HIV-1病毒粒子提供了一种直观地检查感染过程中病毒与靶细胞之间相互作用的方法。然而，现有的区分非特异性内吞的病毒粒子和高效进入宿主细胞细胞质的病毒粒子的方法仍然存在问题。因此，我们检测了一系列膜靶向的荧光融合蛋白构建体被整合到病毒粒子中的能力。我们发现，通过添加c-Src(S15)的N端15个氨基酸序列而定位于质膜的荧光蛋白融合蛋白(S15)有效地包装到HIV病毒粒子中。利用与该序列融合的荧光蛋白，我们产生了S15-mCherry和GFP-VPR双重标记的病毒粒子。重要的是，我们可以检测到融合后S15-mCherry膜信号的丢失。在感染VSV-g假型HIV病毒粒子后，我们发现在感染过程中可测量到特异性的膜标记丢失。当使用巴菲霉素A阻止融合时，不会观察到这种荧光损失。这种对非生产性内吞病毒粒子和那些在感染过程中积极进行步骤的病毒粒子的区分能力的增强，将有助于在显微镜下检查感染的早期步骤。(C)2006 Elsevier Inc.保留所有比赛。
GFP-Vpr labeled HIV-1 virions have provided a method to visually examine the interactions between the virus and target cell during infection. However, existing methods to discriminate between virions that have been non-specifically endocytosed from those that have productively entered the host cell cytoplasm have remained problematic. Therefore, we examined the ability of a series of membrane-targeted fluorescent fusion protein constructs to be incorporated into virions. We find that a fluorescent protein fusion targeted to the plasma membrane by the addition of the N-terminal 15 amino acid sequence of c-Src (S15) is efficiently packaged into HIV virions. Using fluorescent proteins fused to this sequence, we have generated virions dually labeled with S15-mCherry and GFP-Vpr. Importantly, we can detect the loss of this S15-mCherry membrane signal following fusion.After infection with VSV-g pseudotyped HIV virions, we find a measurable, specific loss of membrane label during infection. This loss of fluorescence is not observed when fusion is prevented using bafilomycin A. This increased ability to discriminate between non-productively endocytosed virions and those actively undergoing steps in the infectious process will facilitate efforts to examine early steps in infection microscopically. (c) 2006 Elsevier Inc. All fights reserved.