Dale Benjamin, McNerney Gregory P, Thompson Deanna L, Hübner Wolfgang, Huser Thomas, Chen Benjamin K
Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine.
J Vis Exp. 2010 Oct 7(44):2061. doi: 10.3791/2061.
By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.
通过将绿色荧光蛋白与他们感兴趣的蛋白质融合，生物学家现在能够使用荧光视频显微镜研究复杂的活细胞过程。为了追踪人类免疫缺陷病毒核心蛋白在人类免疫缺陷病毒细胞间传播过程中的运动，我们在一种名为HIV Gag-iGFP的HIV感染性分子克隆中对Gag蛋白进行了绿色荧光蛋白标记。我们使用视频共聚焦显微镜研究这种病毒克隆。在接下来的可视化实验中，我们用HIV Gag-iGFP转染人类T细胞系，并使用荧光标记的未感染CD4+ T细胞作为病毒的靶细胞。使用不同的荧光标记，我们可以很容易地追踪病毒的产生以及其穿过称为病毒突触的细胞间结构的运输过程。简单的透气成像室使我们能够通过实时共聚焦显微镜从几分钟到几天观察突触。这些方法可用于追踪病毒蛋白从一个细胞移动到另一个细胞的过程。
