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 antibodi es and is responsible for the calcium-dependent uncoating of the virus duri ng cell entry. We have purified, characterized, and crystallized recombinan t 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 u ltracentrifugation demonstrate that, in the presence of calcium, purified V P7 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 par ticles. 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 neu tralizing epitopes. (C) 2000 Academic Press.