Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers.
Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes.
Dormitzer
P R
PR
Laboratory of Molecular Medicine, Enders 673, Children's Hospital, 320 Longwood Avenue, Boston, Massachusetts 02115, USA. dormitze@crystal.harvard.edu
Greenberg
H B
HB
Harrison
S C
SC
D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D002122:Calcium Chloride; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D036022:Capsid Proteins; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004492:Edetic Acid; D007313:Insecta; D008253:Macaca mulatta / Q000821:virology; D013058:Mass Spectrometry; D012401:Rotavirus / Q000276:immunology*; D014461:Ultracentrifugation
D000956:Antigens, Viral; D036022:Capsid Proteins; C050779:VP7 protein, Rotavirus; D004492:Edetic Acid; D002122:Calcium Chloride