3-DIMENSIONAL STRUCTURE OF THE ROTAVIRUS HEMAGGLUTININ VP4 BY CRYOELECTRON MICROSCOPY AND DIFFERENCE MAP ANALYSIS

The three-dimensional structure of the rotavirus spike haemagglutinin viral protein 4 (VP4) has been determined to a resolution of 26 Angstrom by cryo-electron microscopy and difference analysis of intact virions and smooth (spikeless) particles. Native and spikeless virions were mixed prior to cryo-preservation so that both structures could be determined from the same micrograph, thereby minimizing systematic errors. This mixing strategy was crucial for difference map analysis since VP4 only accounts for similar to 1% of the virion mass. The VP4 spike is multi-domained and has a radial length of similar to 200 Angstrom with similar to 110 Angstrom projecting from the surface of the virus. Interactions between VP4 and cell surface receptors are facilitated by the bi-lobed head, which allows multi-site interactions, as well as the uniform distribution of the VP4 heads at maximum radius. The bi-lobed head is attached to a square-shaped body formed by two rods that have a slight left-handed helical twist. These rods merge with an angled, rod-like domain connected to a globular base similar to 85 Angstrom in diameter. The anchoring base displays pseudo 6-fold symmetry. This surprising finding may represent a novel folding moth in which a single polypeptide of VP4 contributes similar but non-equivalent domains to form the arms of the hexameric base. The VP4 spike penetrates the virion surface similar to 90 Angstrom and interacts with both outer (VP7) and inner (VP6) capsid proteins. The extensive VP4-VP7 and VP4-VP6 interactions imply a scaffolding function in which VP4 may participate in maintaining precise geometric register between the inner and outer capsids. Finally, we hypothesize that the close interactions between VP4 and VP6 may allow binding of VP4 to immature inner capsid particles in the viroplasm, which then bud through the membrane of the ER to acquire the outer capsid layer that is formed by VP7.