Leopold P L, Kreitzer G, Miyazawa N, Rempel S, Pfister K K, Rodriguez-Boulan E, Crystal R G
Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University-New York Presbyterian Hospital, NY 10021, USA.
Hum Gene Ther. 2000 Jan 1;11(1):151-65. doi: 10.1089/10430340050016238.
Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.
修饰病毒因其能够将遗传物质高效转移至靶细胞核而被用作基因转移载体。为了更好地理解5型腺病毒（Ad）的细胞内转运过程，荧光团被共价连接到Ad衣壳上，并在人肺上皮癌细胞系（A549）感染的最初一小时内观察了荧光Ad在细胞质中的移动情况。Ad转运的特征在于其实现核膜定位的能力以及在细胞质中的定向移动。在对照条件下，Ad感染A549细胞60分钟后可实现高效的核定位，而添加25微摩尔诺考达唑使微管细胞骨架解聚会可逆性地抑制核定位的发展。相比之下，添加1微摩尔细胞松弛素D使微丝解聚对核定位没有影响。对Ad运动性的直接视频观察表明，诺考达唑而非细胞松弛素D会导致Ad在A549细胞质中的快速线性转运出现可逆性降低。显微注射针对微管依赖性运动蛋白胞质动力蛋白而非驱动蛋白的功能阻断抗体可阻断Ad的核定位，这与Ad在有丝分裂纺锤体处积累所表明的净负端定向运动一致。荧光比率成像显示转运中的Ad所处环境为中性pH，从而得出一个模型，即Ad与完整的微管细胞骨架和功能性胞质动力蛋白的相互作用发生在内体逃逸之后，并且是5型腺病毒核定位的必要前提条件。
