Three-dimensional structures of a native simian and reassortant rotavi
rus have been determined by electron cryomicroscopy and computer image
processing. The structural features of the native virus confirm that
the hemagglutinin spike is a dimer of VP4, substantiated by in vivo ra
diolabeling studies. Exchange of native VP4 with a bovine strain equiv
alent results in a poorly infectious reassortant. No VP4 spikes are de
tected in the three-dimensional reconstruction of the reassortant. The
difference map between the two structures reveals a novel large globu
lar domain of VP4 buried within the virion that interacts extensively
with the intermediate shell protein, VP6. Our results suggest that ass
embly of VP4 precedes that of VP7, the major outer shell protein, and
that VP4 may play an important role in the receptor recognition and bu
dding process through the rough endoplasmic reticulum during virus mat
uration.