Bhagwat Amar R, Le Sage Valerie, Lakdawala Seema S
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Methods Mol Biol. 2018;1836:303-327. doi: 10.1007/978-1-4939-8678-1_15.
Influenza viruses exhibit a complex life cycle that is still poorly understood. It involves independent replication of each of the eight segments that make up its genome and subsequent coordinated assembly as they egress from the host cell. Fast, time-resolved volumetric live cell imaging offers a powerful tool for understanding the various host mechanisms hijacked by the virus. Here, we describe the methods necessary for generating influenza viruses that carry a fluorescently tagged polymerase complex, infection of biologically relevant cells with these viruses, and finally protocols for live cell imaging and analysis.
流感病毒呈现出一个仍未被充分理解的复杂生命周期。它涉及构成其基因组的八个片段中的每一个的独立复制，以及随后当它们从宿主细胞中释放时的协调组装。快速、时间分辨的体积活细胞成像为理解病毒劫持的各种宿主机制提供了一个强大的工具。在这里，我们描述了产生携带荧光标记聚合酶复合物的流感病毒、用这些病毒感染生物学相关细胞所需的方法，以及最终活细胞成像和分析的方案。
