FLUORESCENT VIRIONS - DYNAMIC TRACKING OF THE PATHWAY OF ADENOVIRAL GENE-TRANSFER VECTORS IN LIVING CELLS

The pathogenic agent, adenovirus (Ad), has taken on a new role as a ve ctor for gene transfer in both laboratory and clinical settings, To he lp understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1(-), E3(-) Ad vect ors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cel l line including binding, internalization, escape from endosomes, tran slocation to the nucleus, dissociation of capsids and gene expression, The data demonstrate that Ad internalizes with a at(1/2) 2.5 min, bre aks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 mu m/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection, Interes tingly, 24 hr after infection, half of the initially internalized fluo rescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded, Fluor escent labeling of virions provides a novel quantitative, morphologica l strategy to characterize the interaction of gene transfer vectors wi th the intracellular environment.