ABSTRACT
The rotavirus nucleocapsid protein (VP6) is the major structural protein of inner capsid particles (ICP). VP6 is essential for RNA transcription and binds to a virally encoded glycoprotein receptor (NSP4) involved in the rotavirus assembly pathway. To explore the structure of VP6, two-dimensional (2D) crystals of VP6 were generated and examined by electron microscopy and image processing. Fourier transforms computed from low-dose images of negatively stained 2D VP6 crystals displayed complete data to 13 A resolution for p6 plane group symmetry. To correct for the resolution dependent fall-off of the amplitudes derived from electron microscopic images, the rotavirus VP6 amplitudes were scaled to the bluetongue VP7 amplitudes derived from the atomic model by applying a B factor of -360 A-2. The unit cell (a=b=101(+/-2)A, gamma=120(+/-1) degrees) contains two VP6 trimers, each composed of three roughly circular subunits approximately 30 A in diameter. The trimeric organization of VP6 is similar to the oligomeric structure of VP6 when assembled in T=13l icosahedral inner capsid particles at 25 to 40 A resolution. However, a channel at the center of the trimer is better resolved in our map at 15 A resolution. The projection structure of rotavirus VP6 was compared to the homologous protein (VP7) of bluetongue virus, which is also a member of the family of Reoviridae. Notably, both VP6 and bluetongue VP7 assemble as 260 capsomers on the surface of the inner capsid. To compare VP6 and VP7, a projection map of bluetongue VP7 at 15 A resolution was generated using the atomic model derived by X-ray crystallography. VP6 and VP7 both exhibit a trimeric organization with a central channel, even though the alignment identity between the 45 kDa VP6 and the 38 kDa VP7 primary sequences is only 12%. The ability of VP6 to form well-ordered 2D crystals should enable a higher resolution structure analysis by cryo-electron microscopy that will extend our understanding of the icosahedral ICP structure, clarify the mechanism by which VP6 interacts with the NSP4 receptor, and allow a more detailed comparison of VP6 and VP7.
