Hoffmann Anja B, Mazelier Magalie, Léger Psylvia, Lozach Pierre-Yves
From CellNetworks Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
Methods Mol Biol. 2018;1836:159-183. doi: 10.1007/978-1-4939-8678-1_8.
To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.
为了感染宿主细胞，病毒必须进入细胞内区室。感染过程始于病毒附着在细胞表面。随后会发生一系列复杂的事件，这些事件高度动态、紧密交织，且往往难以研究。这包括病毒在质膜上的移位、与受体的结合、信号传导、内化，以及病毒基因组和物质释放到细胞质中。在过去几十年中，灵敏、准确的基于荧光的技术的出现为该领域的研究开辟了新的视角。在固定细胞和活细胞中可视化单个病毒颗粒以及对每个病毒进入步骤进行定量分析已成为可能。在此，我们描述了对病毒颗粒进行荧光标记的程序。我们还举例说明了如何使用这一强大工具，通过诸如高速共聚焦和全内反射显微镜、流式细胞术和荧光测定法等最新的基于荧光的技术来解析病毒的进入过程。
