Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis

Philip L. Leopold, Geri Kreitzer, Naoki Miyazawa, Stephanie Rempel, K. Kevin Pfister, Enrique Rodriguez-Boulan, Ronald G. Crystal

Research output: Contribution to journalArticle

205 Citations (Scopus)

Abstract

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 μM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 μM 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.

Original languageEnglish
Pages (from-to)151-165
Number of pages15
JournalHuman gene therapy
Volume11
Issue number1
DOIs
Publication statusPublished - 24 Jan 2000

    Fingerprint

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Leopold, P. L., Kreitzer, G., Miyazawa, N., Rempel, S., Pfister, K. K., Rodriguez-Boulan, E., & Crystal, R. G. (2000). Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Human gene therapy, 11(1), 151-165. https://doi.org/10.1089/10430340050016238