The C-terminal 88 amino acids of the Sendai virus P protein have multiple functions separable by mutation

The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocap sid (NC) template through a P-NP interaction. To identify P amino acids res ponsible for binding we performed site-directed mutagenesis on the C-termin al 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein -protein interactions. All the mutants formed P oligomers and bound to L pr otein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others-JT4, JT6, and JT9-were complet ely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. N C binding was inhibited by combinations of the inactive mutations. The rema ining P mutants were active in transcription but defective in various aspec ts of genome replication. Some P mutants were defective in NP0 binding and abolished the reconstitution of replication from separate P-L and NP0-P com plexes. In some of these cases the coexpression of the wt polymerase with t he mutant NP0-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occu rred in vivo, but not in vitro, suggesting that the intact cell is providin g an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions bes ides NC binding that can be separated by mutation.