Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
PLoS One. 2011;6(7):e22007. doi: 10.1371/journal.pone.0022007. Epub 2011 Jul 22.
Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches.Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.
荧光标记的人类免疫缺陷病毒（HIV）衍生物，结合先进的荧光显微镜技术的使用，可以直接观察病毒生命周期中的动态事件和各个步骤。用荧光蛋白（FP）标记的 HIV 蛋白已成功用于 HIV-细胞相互作用的活细胞成像分析。然而，FP 在其物理化学性质和成熟动力学方面存在局限性。此外，为了获得适合多色成像设置的光谱变化，需要克隆和表征多个独立的 FP 标记构建体。相比之下，所谓的 SNAP 标签代表一种遗传编码的非荧光标签，它在自标记反应中介导与荧光底物分子的特异性共价偶联。将 SNAP 标签融合到感兴趣的蛋白质上，可以用各种合成染料特异性标记融合蛋白，从而为荧光成像方法提供更大的灵活性。在这里，我们描述了 HIV 衍生物 HIV(SNAP)的构建和表征，该衍生物在病毒结构多蛋白 Gag 内携带 SNAP 标签作为附加结构域。将标签引入 Gag 的基质结构域的 C 末端附近不会干扰颗粒组装、释放或蛋白水解病毒成熟。修饰后的病毒粒子具有感染性，可以在组织培养中繁殖，尽管复制能力降低。SNAP 结构域在 Gag 内的插入允许使用 SNAP 反应性染料特异性染色产生病毒的多蛋白，以及通过随机光学重建显微镜观察单个病毒粒子和病毒出芽部位。因此，HIV(SNAP)是一种多功能工具，它扩大了使用活细胞成像和亚衍射荧光显微镜分析 HIV-细胞相互作用的可能性。
