Reexamination of the Sendai virus P protein domains required for RNA synthesis: A possible supplemental role for the P protein

Abstract

The Sendai virus P protein plays a central role in viral genome amplification and expression, forming complexes with the viral L protein to generate the polymerase (P-L) and unassembled N (P-N°). This latter complex prevents N from selfassembling illegitimately, i.e., independently of the concurrent assembly of a nascent viral genome, and is thought to represent the functional form of N in nucleocapsid assembly. Based upon earlier functional studies using an in vitro transcription/replication system in which the P, L, and N proteins were coexpressed, we identified two regions of the P protein required for RNA synthesis, namely, the C-terminal 40% of the protein, and a second, apparently redundant domain near the N terminus (either amino acids 1-77 or 78-145). The lack of sequence conservation in this second region, apart from overall negative charge, was reminiscent of the acidic activation domains of cellular transcription factors. However, we recently mapped a chaperone domain at the N-terminal of P (aa 33-41), which is required for stable complex formation with unassembled N° and, thus, for assembly and genome replication. In this present study we show that coexpression of N protein with P deletion mutants lacking this region (e.g., P(Δ1-324)) results in the sequestration of the mutant P by the illegitimately assembled form of N. As a consequence, P protein is unavailable for RNA synthesis from bona fide templates. We also find that in the absence of coexpressed N protein, the entire N-terminal 60% of the P protein is not required for mRNA synthesis. During these studies, a supplemental role for the P protein in viral RNA synthesis, independent of stable complex formation with L, was observed. This function involves, at least in part, the binding of additional copies of P to the N RNA template.