Mapping the phosphoprotein binding site on Sendai virus NP protein assembled into nucleocapsids

Abstract To catalyze RNA synthesis, the Sendai virus P–L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP 0 –P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP–RNA and NP–NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401–524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein–protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492–524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472–491, however, abolished replication activity due to a specific defect in the formation of the NP 0 –P complex. Binding of the P protein of the polymerase complex to NC required aa 462–471 of NP, while self-assembly of NP into NC required aa 440–461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.