The overall objective of the program is to understand the complete assembly process of BTV as an example of a complex RNA arbovirus and to define the important morphogenetic events and processes involved in the production of BTV virions. The results and systems that are established will eventually allow us to determine if these events can be selectively inhibited in an infected cell and establish specific means for antiviral therapy. To achieve an understanding of the morphogenetic events in virus assembly the ultrastructure of BTV virions will be analysed by the method of image processing electron micrography. In addition, their composition (in terms of protein types and molar amounts) will be determined based on the precise information of viral protein sizes now available from the cDNA sequences. Subvirus structures obtained during the infection course in vivo (and in vitro formed structures) will be similarly analysed. Available BTV DNA clones will be expressed using available baculovirus expression vectors. (Four BTV genes (L2, L3, M5, S10) have already been individually expressed, yields=10-25% of the total cellular protein.) The expressed products will be used in vitro in a variety of procedures to demonstrate and study protein-protein and protein- nucleic acid interactions that are pertinent to the BTV assembly process. The procedures will include proven binding assays and protein cross-linking studies. It is anticipated that the data, together with the structural analyses, will allow a model of the BTV assembly process to be established. Using new multiple expression vectors combinations of BTV genes will be expressed from different promoters in the same recombinant baculovirus. These recombinants will allow cooperative interactions of those gene products to be examined in vivo, obviating the problems associated with in vitro reactions, and allowing systems to be established (e.g., use of antagonist proteins, peptides, etc.) to test the processes involved in the assembly of BTV components.
|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|