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Journal of Virology, July 2000, p. 6368-6376, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Selective Membrane Permeabilization by the Rotavirus VP5* Protein Is Abrogated by Mutations in an Internal Hydrophobic Domain

William Dowling,1,2,dagger Evgeniya Denisova,1 Rachel LaMonica,1,2,3 and Erich R. Mackow1,2,3,*

Department of Medicine1 and Department of Molecular Genetics and Microbiology,2 SUNY at Stony Brook, Stony Brook, New York, and Northport VA Medical Center, Northport, New York3

Received 21 December 1999/Accepted 17 April 2000

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta -D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta -D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca]i, which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.

* Corresponding author. Mailing address: Departments of Medicine and Microbiology, HSC T17, Rm. 60, SUNY at Stony Brook, Stony Brook, NY 11794-8173. Phone: (631) 444-2120. Fax: (631) 444-8886. E-mail: EMackow{at}mail.som.sunysb.edu.

dagger Present address: Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD 20850.

Journal of Virology, July 2000, p. 6368-6376, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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