Ebola virus VP40-induced particle formation and association with the lipid bilayer.
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.
Jasenosky
L D
LD
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Neumann
G
G
Lukashevich
I
I
Kawaoka
Y
Y
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D029043:Ebolavirus / Q000235:genetics / Q000378:metabolism*; D016563:Filoviridae; D017353:Gene Deletion; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011485:Protein Binding; D012965:Sodium Chloride; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
D008051:Lipid Bilayers; C080981:VP40 protein, virus; D014763:Viral Matrix Proteins; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D017830:Octoxynol; C010615:Nonidet P-40