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Journal of Virology, September 2002, p. 9307-9322, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9307-9322.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Novel Cell Entry Pathway for a DAF-Using Human Enterovirus Is Dependent on Lipid Rafts

Amanda D. Stuart,1* Hannah E. Eustace,1 Thomas A. McKee,2 and T. D. K. Brown1

Division of Virology, Department of Pathology, University of Cambridge, CB2 1QP Cambridge, United Kingdom,1 Institute Universitaire de Pathologie, CH-1011 Lausanne, Switzerland2

Received 16 October 2000/ Accepted 4 June 2002

The glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein decay-accelerating factor (DAF) is used by a number of enteroviruses as a receptor during infection. DAF and other GPI-anchored proteins can be found in cholesterol-rich ordered domains within the plasma membrane that are known as "lipid rafts." We have shown, by using drugs to specifically inhibit various endocytosis routes, that infection by a DAF-using strain of echovirus 11 (EV11) is dependent upon cholesterol and an intact cytoskeleton, whereas a non-DAF-using mutant derived from it was unaffected by these drugs. Using RNA transfection and virus-binding assays, we have shown that this requirement for cholesterol, the actin cytoskeleton, and the microtubule network occurs postbinding of the virus but prior to uncoating of the RNA, indicating a role during virus entry. Confocal microscopy of virus infection supported the role of cholesterol and the cytoskeleton during entry. In addition, [35S]methionine-labeled DAF-using EV11, but not the non-DAF-using EV11, could be copurified with lipid raft components during infection after Triton X-100 extraction. These data indicate that DAF usage by EV11 enables the virus to associate with lipid rafts and enter cells through this novel route.

* Corresponding author. Mailing address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Rd., CB2 1QP Cambridge, United Kingdom. Phone: 44-1223-336918. Fax: 44-1223-336926. E-mail: ads35{at}mole.bio.cam.ac.uk.

Journal of Virology, September 2002, p. 9307-9322, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9307-9322.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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