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Journal of Virology, August 2001, p. 7339-7350, Vol. 75, No. 16
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
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
-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.
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