The overall objectives of the program is to understand the assembly process of bluetongue virus (BTV) as an example of a complex non- enveloped RNA arbovirus by defining the essential morphogenetic interactions of viral structural proteins and the determinants that are involved for intra- and inter-molecular protein-protein interactions in the assembly and morphology of the particles. To achieve an understanding of the morphogenetic events in BTV assembly, use will be made of an established baculovirus multiple protein expression system in combination with recent data obtained from the three-dimensional structure of particles (eg, cryo-electron microscopy) and the X-ray crystallographic structure of the major virion protein VP7, 260 trimers of which are the building blocks of the icosahedral core. The baculovirus expression system allows us to produce and manipulate particulate structural entities representing BTV, eg, core-like particles (CLPs) comprising VP3 and VP7, and virus-like particles (VPLs) comprising VP2, VP3, VP5 and VP7. How the trimeric units are formed and generate a complex lattice of icosahedral surface of the core, or how VP3 and VP7 trimers assemble into CLPs and VP2 and VP5 interact with such cores to form VLPs will be determined. Deletion, site-specific and domain switched mutant proteins will be generated to identify the motifs relevant to the BTV assemble process. In addition, high resolution (22A) 3-dimensional ultra-structural analysis of various baculovirus synthesized CLPs will be used to define the topography and organization of VP3 subcores and the three innermost proteins (the minor proteins, VP1, VP4 and VP6) that are encapsidated within the core and CLPs.
| Hassan, S H; Wirblich, C; Forzan, M et al. (2001) Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization. J Virol 75:8356-67 |
