Rabies virus ribonucleoprotein (RNP) complex which is comprised of the genomic negative strand RNA surrounded by nucleoprotein (N), phosphoprotein (NS), and RNA polymerase (L), is capable of initiating infection in cells, and is therefore the necessary unit for both transcription and replication. The N and NS proteins are the major components of the RNP complex, and are thought to play important roles in the process of transcription and replication of viral RNA by interacting with each other, as well as with L protein and viral RNA. As a first step toward a better understanding of rabies virus transcription and replication, I propose to study the structure of rabies virus N protein and its interaction with NS protein and genomic RNA, and to locate the regions on N protein that interact with these components. The N and NS proteins of rabies virus ERA strain will be expressed in insect cells, using baculovirus vectors, and purified individually or as complexes. The detailed structure of these proteins or protein complexes will be studied by crystallization and X-ray diffraction. The interaction of N and NS proteins will be studied by mixing the purified N and NS proteins at different ratios and analyzing the complexes formed. The interaction of N protein with viral RNA will be investigated by assessing the ability of expressed N protein to encapsidate in vitro synthesized rabies virus leader RNA, and thereby protect it from ribonuclease digestion. The interaction between rabies virus N and NS proteins and between N protein and genomic RNA will also be studied by simultaneously expressing the components in the newly available one step in vitro transcription/translation system. The regions of these components (N protein, NS protein, genomic RNA) that are involved in the interaction will also be identified by expressing protein mutants with C-terminal truncations and internal deletions, and studying the binding activity of these mutagenized proteins.
