Jouvenet Nolwenn, Bieniasz Paul D, Simon Sanford M
Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York 10065, USA.
Nature. 2008 Jul 10;454(7201):236-40. doi: 10.1038/nature06998. Epub 2008 May 25.
Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport. However, the assembly of individual virions has never been observed in real time. Insights into this process have come primarily from biochemical analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial. Here we describe quantitatively the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivatives of Gag, the major structural component of HIV-1-which is sufficient to drive the assembly of virus-like particles-with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5-6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the determination of parameters of viral assembly that are inaccessible with conventional techniques.
对活细胞中单个病毒粒子的观察已促成了对其附着、进入和细胞内运输的特性描述。然而，单个病毒粒子的组装过程从未被实时观察到。对这一过程的深入了解主要来自对病毒粒子群体的生化分析或对固定感染细胞的显微镜研究。因此，一些组装特性，如动力学和位置，要么未知，要么存在争议。在这里，我们实时定量描述了单个病毒粒子的产生过程，从组装开始到出芽和释放。我们使用荧光共振能量转移、光漂白后荧光恢复和活细胞中的全内反射荧光显微镜，研究了HIV-1主要结构成分Gag的荧光标记衍生物，该衍生物足以驱动病毒样颗粒的组装。病毒粒子在质膜上单个出现，随着Gag蛋白在细胞中积累，其组装速率加快，完成单个病毒粒子的组装通常需要5 - 6分钟。这些方法使我们能够以前所未有的视角观察单个病毒粒子的产生过程，并确定传统技术无法获得的病毒组装参数。
