Fluorescent Virions: Dynamic Tracking of the Pathway of Adenoviral Gene Transfer Vectors in Living Cells

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 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 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. Gene transfer vectors based on replication-deficient adenovirus are exemplary in that they exhibit highly efficient transfer of genome into cells, through the cytoplasm, and into the nucleus. The development of future gene transfer vectors will likely benefit from a detailed understanding of the mechanism of adenoviral infection. To improve our understanding of this process, new probes that can report the intracellular position of adenovirus capsids will be useful. The development of fluorescently conjugated adenovirus capsids enables high-resolution studies of adenoviral infection pathways in the living cells.