We are studying molecular mechanisms of RNA synthesis by Sendai virus, a useful laboratory model of the medically important paramyxovirus family. Structure-function relationships of the three virus nucleocapsid proteins, L, P and NP, will be explored by site-specific mutagenesis of recombinant DNA molecules representing the genes that specify these proteins. Each mutated gene will be transfected into eukaryotic cells, where the protein it expresses can be complemented by a virus mutant nonconditionally defective in the same gene. NP protein functions in question are assembly into nucleocapsids, interactions within the nucleocapsid with viral and RNA and the P and L protein, interactions with the viral envelope proteins that culminate in virus budding, and regulation of virus RNA replication. The auxiliary nucleocapsid proteins, P and L, cooperate to synthesize viral RNA;
we aim to determine which species possesses the catalytic site for each of the enzymatic activities involved in 5'-terminal modifications of viral transcripts and map the amino acids that make up those sites. We will also map sites involved in P-L interactions and the assembly of P and L into nucleocapsids, and we will identify the functions of the nonstructural C proteins whose genetic elements are embedded within the P gene. We propose an in vitro system for generating viral RNA species from recombinant DNA, enabling us to identify the nucleation site for DNA encapsidation and to generate artificial deletion mutant (DI) viral nucleocapsids containing nucleotide sequences that regulate replicative and transcriptional RNA synthesis. We plan to define the regulatory roles of the nucleotide in these sequences by introducing site-specific mutations into recombinant DNA molecules that encode them. We will then introduce DI nucleocapsids made from the DNA templates into eukaryotic cells infected with nondefective helper virus, and assess the effects of the mutations on RNA synthesis specified by the DI genomes.