Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University , 313 Ferst Drive, UA Whitaker Bldg, Atlanta, Georgia 30332, United States.
ACS Nano. 2014 Jan 28;8(1):302-15. doi: 10.1021/nn405998v. Epub 2013 Dec 23.
The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses.
荧光标记病毒的创建目前受到成像观察时间（例如，标记包膜蛋白）和病毒感染性恢复（例如，编码 GFP 蛋白）的限制。通过将单个分子敏感的 RNA 杂交探针递送至感染细胞的细胞质中，我们能够分离出单个、感染性、荧光标记的人呼吸道合胞病毒病毒粒子。这一方法在不影响病毒 mRNA 表达、病毒蛋白表达或感染性的情况下实现。测量包括使用 dSTORM 对单个病毒粒子中的病毒蛋白和基因组 RNA 进行表征，开发 GFP 融合测定法，以及开发用于病毒 RNA 产生的脉冲追踪测定法，该方法允许在感染后指定时间检测初始病毒 RNA 和新生 RNA 的产生。活细胞测量包括丝状病毒粒子融合的成像和表征，以及在八小时内对同一细胞内病毒复制的定量。使用探针标记的病毒，可以在亚衍射限制分辨率下对单个病毒颗粒进行表征，并可以在整个复制周期内对单个细胞中的病毒感染进行定量。这一发展的意义在于，在病毒组装过程中对病毒 RNA 进行 MTRIP 标记有可能成为标记和研究许多重要 RNA 病毒的通用方法。
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