211. Novel Fiber-Mediated Adenovirus Interactions in Primary Rabbit Lacrimal Acini

Abstract

Top of pageAbstract Purpose: Cellular infection by adenovirus serotype 5 (Ad5) is thought to be mediated by fiber capsid and coxsackie-adenovirus receptor (CAR) binding followed by the interaction of penton capsid with |[alpha]|v integrins, which triggers clathrin-mediated endocytosis. The purpose of this study was to identify the capsid proteins responsible for mediating Ad5 entry in cultured lacrimal acinar epithelial cells, the major source of tear proteins released into ocular surface fluid. Methods: Rabbit lacrimal gland acinar cells were isolated and cultured for 2 - 3 d. To examine the mechanism of entry and the intracellular trafficking of Ad5 and its capsids, reconstituted acini were exposed to Ad-LacZ (MOI=5-20), recombinant penton (20 |[mu]|g/ml) or knob protein (20 |[mu]|g/ml) at 37|[deg]| for short time intervals (0-120 m) and for 16-18 h. Their distributions were evaluated by confocal fluorescence microscopy or a trypsinization-based biochemical assay. To determine the identity of capsids and their binding partners responsible for mediating Ad5 entry, acinar cells were preincubated with various doses of penton, fiber, knob, or penton-blocking peptides, RGD or LDV, for 2 h at 4|[deg]|. Ad-GFP (MOI=2) was then added and incubated for 1 h at 4|[deg]| before incubating cells for 10-12 h at 37|[deg]|. Transduction efficiency was assayed by GFP positivity, determined by FACS analysis. To determine the involvement of heparin sulfate glycosaminoglycan (HS-GAG), in some experiments Ad-GFP was preincubated with heparin at 37|[deg]| for 1 h before being added to the cells. Results: Ad5 showed pronounced intracellular punctate staining after 30 m incubation and retained an intracellular labeling pattern by 2 h that was comparable to that seen with overnight exposure. Penton remained associated with the plasma membrane after acute and chronic exposure, while sustained knob exposure resulted in a comparable intracellular labeling pattern to that seen for Ad5. Fiber significantly (p|[le]|0.05) and substantially inhibited Ad5 infectivity (17.42|[plusmn]|3.53% of control, n=4) while penton had no effect (102.68|[plusmn]|9.63% of control, n=7). Knob also induced a comparable inhibitory effect on Ad5 infectivity (17.23|[plusmn]|3.39% of control, n=8) relative to fiber while RGD and LDV had no effect. Lower doses of fiber and knob also exhibited an inhibitory effect which was not as potent. However, CAR expression appeared to be minimal in acinar cells compared to other cells. Heparin had a modest but significant inhibitory effect on Ad5 infectivity which was additive with that of knob. Conclusions:These studies suggest that Ad5 internalization into lacrimal acini is through a unique fiber-dependent pathway rather than the consensus penton-mediated mechanism seen in simple cell models. HS-GAGs may serve as cooperative receptors for Ad5 by increasing the efficiency of the binding and infection mediated by CAR or another fiber receptor. Further characterization of the nature of virus-cell interactions will be important in consideration of the toxicity and efficacy of Ad5 capsids for non-viral gene delivery.