Fluorescent virions: Dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells

Philip L. Leopold, Barbara Ferris, Irene Grinberg, Stefan Worgall, Neil R. Hackett, Ronald Crystal

Research output: Contribution to journalArticle

196 Citations (Scopus)

Abstract

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 El-, 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 t(1/2) 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 μm/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.

Original languageEnglish
Pages (from-to)367-378
Number of pages12
JournalHuman Gene Therapy
Volume9
Issue number3
DOIs
Publication statusPublished - 10 Feb 1998
Externally publishedYes

Fingerprint

Adenoviridae
Virion
Endosomes
Genes
Capsid
Infection
Fluorescence Microscopy
Fluorescence
Epithelial Cells
Gene Expression
Cell Line
Lung

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Fluorescent virions : Dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells. / Leopold, Philip L.; Ferris, Barbara; Grinberg, Irene; Worgall, Stefan; Hackett, Neil R.; Crystal, Ronald.

In: Human Gene Therapy, Vol. 9, No. 3, 10.02.1998, p. 367-378.

Research output: Contribution to journalArticle

Leopold, Philip L. ; Ferris, Barbara ; Grinberg, Irene ; Worgall, Stefan ; Hackett, Neil R. ; Crystal, Ronald. / Fluorescent virions : Dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells. In: Human Gene Therapy. 1998 ; Vol. 9, No. 3. pp. 367-378.
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