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Journal of Virology, April 1999, p. 3147-3153, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rotavirus Capsid Protein VP5* Permeabilizes
Membranes
Evgeniya
Denisova,1,2
William
Dowling,1,2
Rachel
LaMonica,1,2
Robert
Shaw,1,3
Suzanne
Scarlata,4
Franco
Ruggeri,5 and
Erich R.
Mackow1,2,3,*
Department of
Medicine,1 Department of Molecular
Genetics and Microbiology,2 and
Department of Physiology and
Biophysics,4 SUNY at Stony Brook, Stony Brook,
and Northport VA Medical Center,
Northport,3 New York, and Instituto
Superiore Di Sanita, Rome, Italy5
Received 29 July 1998/Accepted 29 December 1998
Proteolytic cleavage of the VP4 outer capsid spike protein into
VP8* and VP5* proteins is required for rotavirus infectivity and for
rotavirus-induced membrane permeability. In this study we addressed the
function of the VP5* cleavage fragment in permeabilizing membranes.
Expressed VP5* and truncated VP5* proteins were purified by nickel
affinity chromatography and assayed for their ability to permeabilize
large unilamellar vesicles (LUVs) preloaded with carboxyfluorescein
(CF). VP5* and VP5* truncations, but not VP4 or VP8*, permeabilized
LUVs as measured by fluorescence dequenching of released CF. Similar to
virus-induced CF release, VP5*-induced CF release was concentration and
temperature dependent, with a pH optimum of 7.35 at 37°C, but
independent of the presence of divalent cations or cholesterol.
VP5*-induced permeability was completely inhibited by VP5*-specific
neutralizing monoclonal antibodies (2G4, M2, or M7) which recognize
conformational epitopes on VP5* but was not inhibited by VP8*-specific
neutralizing antibodies. In addition, N-terminal and C-terminal
VP5* truncations including residues 265 to 474 are capable of
permeabilizing LUVs. These findings demonstrate that VP5* permeabilizes
membranes in the absence of other rotavirus proteins and that
membrane-permeabilizing VP5* truncations contain the putative fusion
region within predicted virion surface domains. The ability of
recombinant expressed VP5* to permeabilize membranes should
permit us to functionally define requirements for VP5*-membrane
interactions. These findings indicate that VP5* is a
specific membrane-permeabilizing capsid protein which is likely to play
a role in the cellular entry of rotaviruses.
*
Corresponding author. Mailing address: Departments of
Medicine and Microbiology, HSC T17, Rm. 60, SUNY at Stony Brook, Stony Brook, NY 11794-8173. Phone: (516) 444-2120. Fax: (516) 444-8886. E-mail: EMackow{at}mail.som.sunysb.edu.
Journal of Virology, April 1999, p. 3147-3153, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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