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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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