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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.
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