Leopold P L, Ferris B, Grinberg I, Worgall S, Hackett N R, Crystal R G
Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, NY 10021, USA.
Hum Gene Ther. 1998 Feb 10;9(3):367-78. doi: 10.1089/hum.1998.9.3-367.
The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.
病原体腺病毒（Ad）在实验室和临床环境中已成为基因转移的一种新载体。为了帮助理解Ad基因转移载体的细胞内途径和命运，我们将荧光团共价连接到E1 -、E3 - Ad载体上，并使用定量荧光显微镜来评估Ad载体基因转移至A549人肺上皮细胞系的关键步骤，包括结合、内化、从内体逃逸、转运至细胞核、衣壳解离和基因表达。数据表明，Ad以内化半衰期2.5分钟内化，在内体 - 内体融合之前可能就早早从内体中逸出，表现出持续的细胞内平均速度为0.58微米/秒，并且在感染后60分钟内，超过80%内化的荧光团转运至细胞核。有趣的是，感染24小时后，检测到一半最初内化的荧光，但缺乏核定位，这表明衣壳从细胞核中释放出来并可能被降解。病毒粒子的荧光标记提供了一种新颖的定量、形态学策略，用于表征基因转移载体与细胞内环境的相互作用。
