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Journal of Virology, August 2001, p. 7339-7350, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7339-7350.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Proteolysis of Monomeric Recombinant Rotavirus VP4 Yields an Oligomeric VP5* Core

Philip R. Dormitzer,1,* Harry B. Greenberg,2 and Stephen C. Harrison1,3

Laboratory of Molecular Medicine, Children's Hospital, Boston, Massachusetts 021151; Departments of Microbiology and Immunology and of Medicine, Stanford University School of Medicine, Stanford, California 94305; the VA Palo Alto Health Care System, Palo Alto, California 943042; and Howard Hughes Medical Institute and the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 021383

Received 29 January 2001/Accepted 16 May 2001

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta -sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.

* Corresponding author. Mailing address: Laboratory of Molecular Medicine, Enders 673, Children's Hospital, 320 Longwood Ave., Boston, MA 02115. Phone: (617) 355-4795. Fax: (617) 738-0184. E-mail: dormitze{at}crystal.harvard.edu.

Journal of Virology, August 2001, p. 7339-7350, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7339-7350.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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