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 microm/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.

PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles

“…11,14 In the present study, we demonstrate that the PCI method enables increased gene expression compared to conventional adenovirus infection in all six cell lines examined. The efficiency of the PCI treatment varies between the cell lines, with a maximum of 13-fold increase in transgene expression after photochemical treatment.…”

“…[9][10][11][12][13] The enhanced adenoviral gene expression observed after photochemical treatment was surprising considering existing literature stating the adenoviral uptake mechanism to be very efficient, and that up to 90% of the endocytosed adenoviral particles are translocated into the cytosol. 11,14 In the present work, we investigate the role of the adenoviral receptor and examine the endocytic pathway involved during photochemical transduction. The data presented indicate that the adenoviral particles during photochemical transduction use the same cell surface receptors and also follow the same route of internalization as in conventional infection.…”

Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

“…The Alexa Fluor 555 carboxylic acid, succinimidyl ester (Invitrogen) was conjugated to our replication-deficient Ad5 stock using a protocol similar to that described previously (Leopold et al, 1998). Briefly, 100 l of DMSO was added to 1 mg of the lyophilized Alexa Fluor dye.…”

“…The dialyzed Ad-AF555 was adjusted to a final concentration of 30% glycerol and stored at Ϫ20°C in 20-l aliquots. The final concentration of Alexa Fluor 555 dye was determined as described previously (Leopold et al, 1998). We refer to the labeled virus as Ad-AF555.…”

Oncolytic viral therapies

“…[72][73][74][75][76][77][78] Receptor binding leads to internalization of the virus by the cell through receptormediated endocytosis, lysis of the endosomal membrane, escape to the cytosol, trafficking along microtubules towards the nucleus, binding the nuclear envelope, and insertion of the viral genome into the nucleus. [79][80][81][82][83][84][85][86][87][88] The adenoviral reproductive cycle is a highly orchestrated process, and gene modification strategies, which enable selectivity, often reduce replication capacity. At the molecular level, completion of the infectious cycle relies on timely expression of a set of viral regulatory proteins that interact with essential endogenous cellular pathways that determine cell viability.…”