The overall objective of the program is to understand the complete assembly process of BTV as an example of a complex non-enveloped RNA arbovirus by defining the essential morphogenetic interactions of viral components (7 structural proteins, VP1-7 and the viral genome) and how the viral non-structural proteins (three, NS1-3) are involved in the BTV replication process. To achieve an understanding of the morphogenetic events in virus assembly, use will be made of an established baculovirus multiple protein expression system. The system allows us to produce and manipulate particulate structural entities representing BTV (eg, core-like particles.(CLPs) comprising VP3 and VP7, and virus-like articles (VLPs) comprising VP2, VP3, VP5 and VP7). How VP3 and VP7 proteins assemble into CLPs and VP2 and VP5 interact with such cores to form VLPs or how the minor proteins (VP1, VP4 and VP6) interact with the structures will be determined. Deletion, site-specific and domain switched mutant proteins will be generated to identify the motifs relevant to protein-protein interactions relevant to the BTV assembly process and their effect on the intrinsic functions (ss, dsRNA binding, GTP binding, RNA polymerase activity, tubule and inclusion body formation). The 3-dimensional ultra-structural analyses of normal and mutant particles will be undertaken. Procedures will be developed to encapsidate the viral genome and establish a viral RNA replication system.
| 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 |
