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Journal of Virology, September 2004, p. 10122-10132, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.10122-10132.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cytoplasmic Dynein Mediates Adenovirus Binding to Microtubules

Samir A. Kelkar,1,2 K. Kevin Pfister,3 Ronald G. Crystal,1 and Philip L. Leopold1*

Department of Genetic Medicine,1 Graduate Program in Physiology and Biophysics and Systems Biology, Weill Medical College of Cornell University, New York, New York,2 Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia3

Received 29 September 2003/ Accepted 27 April 2004

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% ± 3.5% to 80.7% ± 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.

* Corresponding author. Mailing address: Weill Medical College of Cornell University, Department of Genetic Medicine, 515 E. 71st St., S-1000, New York, NY 10021. Phone: (212) 746-2258. Fax: (212) 746-8383. E-mail: geneticmedicine{at}med.cornell.edu.

Journal of Virology, September 2004, p. 10122-10132, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.10122-10132.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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