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Journal of Virology, December 2006, p. 11833-11851, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.00857-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in Lacrimal Acini

Departments of Pharmacology and Pharmaceutical Sciences,¹ Physiology and Biophysics,² Ophthalmology,³ Cell and Neurobiology, University of Southern California, Los Angeles,⁴ Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, California⁵

Received 26 April 2006/ Accepted 11 September 2006

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to _v integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.

Published ahead of print on 20 September 2006.

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