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Journal of Virology, June 2001, p. 5205-5214, Vol. 75, No. 11
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.11.5205-5214.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Ebola Virus VP40-Induced Particle Formation and Association with the Lipid Bilayer

Luke D. Jasenosky,1 Gabriele Neumann,1 Igor Lukashevich,2,dagger and Yoshihiro Kawaoka1,3,*

Department of Pathobiological Sciences, School of Veterinary Medicine,1 and Department of Pathology and Laboratory Medicine, School of Medicine,2 University of Wisconsin---Madison, Madison, Wisconsin 53706, and Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan3

Received 14 November 2000/Accepted 19 March 2001

Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin---Madison, 2015 Linden Dr. West, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail: kawaokay{at}svm.vetmed.wisc.edu.

dagger Present address: Institute of Human Virology, University of Maryland, Baltimore, MD 21201.

Journal of Virology, June 2001, p. 5205-5214, Vol. 75, No. 11
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.11.5205-5214.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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